Standardized plant extract, process for obtaining the same and uses thereof

ABSTRACT

The present invention refers to a process for obtaining a standardized extract having antinociceptive, anti-inflammatory and antipyretic properties, from at least one part of a plant of genus  Aleurites . Furthermore, the present invention provides a pharmaceutical composition comprising an active ingredient of a pharmaceutically efficient quantity of standardized extract from at least one part of the plant of genus  Aleurites . Finally, the present invention describes a method of treatment and use of the said extract, isolated or in a pharmaceutical composition, for the prevention, control or treatment of painful, inflammatory or febrile affections.

FIELD OF THE INVENTION

The present invention refers to a process for obtaining a standardizedextract having antinociceptive, anti-inflammatory and antipyreticproperties, from a plant of genus Aleurites. The invention furtherprovides a pharmaceutical composition comprising the said standardizedextract, methods of treatment, prevention or control of painful,inflammatory or febrile affections and uses of the said extract.

BACKGROUND OF THE INVENTION

Pain is the most common symptomatic manifestation in humans. It is acomplex condition, often of undefined aetiology. While acute pain isoften the result of traumatism, visceral dyskinesia, acute inflammatoryprocesses and infections, chronic pains are normally related tomusculoskeletal, neuropathic and oncolytic affections and chronicinflammatory processes.

Several mechanisms are related to the occurrence of acute pains:traumatism of the musculoskeletal structures and soft tissues,dysfunctional anomalies, acute inflammatory processes, abdominal,thoracic or oropharyngeal visceral infections and dyskinesias may alltrigger physical, mechanical, thermal and/or chemical stimuli thatactivate the nociceptors in the fibres of the peripheral nervous system.These transmit nociceptive information to the spinal medulla andsubsequently to the hypothalamus and cortical areas involved in theprocessing of sensibility and originating in pain.

The more common chronic manifestations of pain are linked to chronicinflammatory processes normally seen in osteoarthritis, tendonitis,bursitis, spondylitis and other generally autoimmune degenerativeprocesses. More than the actual pain, this process focuses thephenomenon of mechanical and thermal hyperalgesia related to decreasednociceptor thresholds which produce hypersensitivity to movement,mechanical deformation and heat. Hyperalgesia is caused by the action ofnumerous inflammatory mediators released in the painful regions, andmore specifically bradykinin, acetylcholine, histamine and thecytokines, such as interleukins and tumour necrosis factors. Theresulting hypersensitivity justifies the fact that inflamed tissues arepain free when at rest but become painful when stimulated.

The treatment and adequate control of pain and frequent inflammation isby pharmacotherapy, whereby several pharmacologic classes known in thestate of the art are used for analgesic and anti-inflammatory purposes,with a large majority of these being obtained by means of chemicalsynthesis.

The most currently used analgesic and anti-inflammatory drugs are fromthe class of non-steroidal anti-inflammatory drugs (NSAID), such assalyclates, acetic acid derivatives, indolic derivatives and enol acidderivatives, amongst others.

However, the main problem related to the use of the anti-inflammatoriesspecified above is due to their collateral effects, especially duringprolonged administration. These effects include gastritis, ulceration,dyspepsia, nausea, vomiting, allergies, renal insufficiency,irreversible nephropathy, Reye's Syndrome, metabolic acidosis,hypertension and a risk of cardiovascular diseases.

In the case of non-selective NSAIDs, the non-specific inhibitors of thecyclooxygenase (COX) enzyme inhibit both COX-1, considered constitutiveand responsible for the homeostasis of organs such as the stomach andkidneys, as well as COX-2, considered inductive and increasing in theorganism at the genesis of the inflammatory process. COX-2 isresponsible for the production of pro-inflammatory prostanoid mediatorssuch as certain prostaglandin and leukotrienes. The inhibition of thisenzyme (COX-2) is important for the reduction of the inflammatoryprocess. However, the inhibition of COX-1 triggers the adverse effectsdescribed above and, more specifically, the gastrointestinal disordersbecause it is an important regulator of hydrochloric acid production aswell as the production of the bicarbonate and mucous coating protectingthe stomach from the acids and enzymes in the gastric juice.

The use of NSAIDs has been associated with serious complications. Suchproblems either impede adhering to the pharmacologic treatment per se,or render the treatment of chronic painful and/or inflammatory processesimpossible.

In view of these limitations, it is understandable that the developmentof safer therapies or, in other words, with a better risk-benefit ratio,for the treatment of pain and inflammation is necessary, especiallyconsidering the risks associated to the pharmaceuticals currently inuse.

In this context, phytotherapy presents itself as an appropriatealternative to the predominantly synthetic therapeutic arsenal.

The importance and chemical potentiality of medicinal plants can beascertained from scientific research data results whereby approximately500 of the medicines used clinically worldwide originate from naturalproducts and derivatives (Gurib-Fakim, A., 2006, Mol. aspects med., 27,1-93).

Frequently, the pharmacologic effects of phytotherapeutic medicines doesnot occur through the action of a single compound but rather through thecombined activity of the various active substances contained orassociated to it. Generally, the active substance isolated presents adifferent activity or lesser potency than that presented by thephytocomplex.

Contrary to medicines of synthetic origin, the active substances ofphytotherapeutic medicines are almost always found in low concentrationswhich, generally, result in a lower incidence of adverse effects.

Despite the limitations encountered when obtaining, purifying andidentifying natural products, recent technological advances have enabledobtaining more powerful and enhanced products when compared to syntheticproducts. (Koehn, F. E. & Carter, G. T., 2005, Nature, 4, 206-220).

A common issue to the use and production of phytomedicines relate to thevariations in the concentration(s) of the active ingredient(s) which aregenerally secondary metabolites. These metabolites represent aninterface between the plants and their environment and their synthesisis frequently affected by ambient conditions (such as seasonality,circadian rhythm, plant development stage, plant age, temperature,availability of water, ultraviolet (UV) radiation, soil nutrients,altitude, atmospheric composition and plant tissue damage). Suchfactors, as well as others such as gathering conditions, stabilisation,storage and industrial processing may all have an effect on the contentof the secondary metabolites of medicinal plants and thus may also havea major influence on the quality of the phytotherapeutics beingprepared. Therefore, apart from quality control involving modernanalytic techniques and the standardisation of the intermediaryproducts, the source and quality of the raw materials have apreponderant role for obtaining products of constantly reliablecomposition and reproducible therapeutic properties (Gobbo-Neto, L. &Lopes, N. P., 2007, Quím. Nova, 30, 374-381).

The Euphorbiaceae family originated from tropical Asia and the Pacificislands that consist of herbaceous plants, shrubs or trees generallyproducing ligneous latex. In Brazil, they are found in tropical areasbeing widely cultivated in coastal regions but may also be found intemperate regions. This family includes approximately 7.000 species with317 genus (Webster, G. L., 1994, Annals of the Missouri. Botan. Garden,81, 1, 3-32). Former studies of this family revealed a predominantpresence of lipids, terpenoids, alkaloids and hydrocarbons (Hui, W. H. &Hoi, C. T., 1968, Aust. J. Chem., 21, 1675-7). Other studies also showedthe importance of this family as food and in medicines as well as inindustry (Gneco, S. et al., 1996, Bol. Soc. Chil. Quim., 41, 229-233;Villalobos, M. J. P. & astellanos, E. C., 1992, Grasas Y aceites, 43, 1,39-44).

Among the several genus of the Euphorbiaceae family, the followingshould be mentioned being of the most importance due to their diversity,applications and commercial interest: Euphorbia, Croton, Phyllanthus,Jatropha, Sapium, Ricinus, Aleurites (Webster, G. L., 1994, Annals ofthe Missouri. Botan. Garden, 81, 1, 3-32). The genus Aleurites issubdivided in A. trisperma, A. cordata, A. montana, A. fordii, A.montance, A. rockinghamensis, apart form A. moluccana (Villalobos, M. J.P. & Castellanos, E. C., 1992, Grasas y aceites, 43, 1, 39-44; Misra, D.R. e Khastgir, H. N., 1970, Tetrahedron, 26, 12, 3017-3021; Cruz, G. L.Dicionário das plantas úteis do Brasil. 4th ed. Rio de Janeiro:Bertrand, 1964; Pio Correia, M. Dicionário das plantas úteis do Brasil edas exóticas cultivadas. IBDF, Brasília-DF (1984) V:294-295; Fozdar, B.I. et al., 1989, Phytochem., 28, 9, 2459-2461; Agarwal, R. et al., 1995,Fett. Wissench. Technologie-Fat Science Technol., 97, 526-527; Forster,P. I., 1996, Muelleria, 9, 5-13).

The Aleurites moluccana L. Willd., having the synonyms Aleurites trilobaJ. R. & G. Forst, Croton moluccanus L., Jatropha moluccana is an exotictree native of Indonesia having been broadly introduced to Brazil andspreading from the state of São Paulo through to the state of Rio Grandedo Sul, being especially abundant in the state of Santa Catarina. It ispopularly known locally as “Nogueira-de-Iguape”, “Noz-da-India”,“Nogueira-da-India” “Nogueira-de-Bancul”, or simply “Nogueira” (Duke, J.A., Handbook of medicinal herbs. U.S.A.: CRC Press, 1991).

The use of Aleurites moluccana in traditional popular remedies is vastas is the case of most medicinal plants. It is used to control and as anempirical treatment for the following diseases or symptoms: fever,inflammations, asthma, conjunctivitis, hepatitis, headaches,ulcerations, diarrhoea, gonorrhoea and is also used against tumours, asa laxative stimulant, diaphoretic and anti-rheumatic. (Pio Correia, M.Dicionário das plantas úteis do Brasil e das exóticas cultivadas. IBDF,Brasília-DF, 1984, 294-295; Villalobos, M. J. P. & Castellanos, E. C.,1992, Grasas Y aceites, 43, 1, 39-44; Forster, P. I., 1996, Muelleria,9, 5-13; Duke, James A. Handbook of medicinal herbs. U.S.A.: CRC Press,1991; Stuppy, W. et al., 1999, Blumea, 44, 1, 73-98; Locher, C. P. etal., 1995, J. Ethnopharmacol., 49, 23; Hope, B. E. et al., 1993, HawaiiMed., 56, 6, 160-166). Nevertheless, very few studies able tosystematically justify this broad therapeutic potential are described inthe literature.

Certain prior studies by a Belgian research group demonstrated that theextracts of a plant collected in Hawaii showed antiviral activity, morespecifically against the HIV virus (Locher, C. P. et al., 1996,Phytomedicine, 2, 259), as well as an antibacterial effect againstStaphylococus aureus and Pseudomonas aeruginosa (Locher, C. P. et al.,1995, J. Ethnopharmacol., 49, 23).

Other preliminary studies conducted by the present inventors and theircollaborators further demonstrated that non-standard hydroalcoholicextracts of Aleurites moluccana as well as their hexanic fractions,presented analgesic potential in an acetic acid-induced pain model inmice. Furthermore, analysis of the active ingredients isolated led tothe identification of n-hentriacontane, alpha-amyrin, beta-amyrin,alpha-amirinone, beta-amirinone, stigmasterol, beta-sitosterol,campesterol, acetil aleuritolic acid (AAA), swertisin and2″-O-rhamnosylswertisin, with some of these substances havingsignificantly inhibited the acetic acid induced abdominal contortions inmice (Meyre-Silva, C. et al., 1998, Phytomedic., 5, 2, 109-113;Meyre-Silva, C. et al., 1999, Planta Med., 65, 3, 293-294).

In 1999, the inventors and their collaborators also demonstrated thatswertisin, a flavonoid isolated from the leaves of Aleurites moluccana,did not have an analgesic effect with the acetic acid induced abdominalcontortion model in mice. However, its derivate 2″-O-rhamnosylswertisinpresented analgesia approximately 16 times more powerful than aspirinwith this model, thus suggesting that the ramnosil group has animportant role in the analgesic action of these compounds (Meyre-Silva,C. et al., 1999, Planta Med., 65, 3, 293-294).

Thus, former studies conducted by the inventors and their collaboratorssuccessfully identified potential use for the species in the developmentof new phytotherapeutic medicines.

The studies and research hereby presented by the inventors demonstratenew antinociceptive, anti-inflammatory and antipyretic activitiesrelated to the extracts of A. moluccana which are confirmed in the dataand tests described below in the present report.

The inventors further identified a set of remarkable procedures,practices and conditions when compared to the current state of the art,as described below, that contribute to the use of Aleurites moluccana asa medicine/pharmaceutical input by guaranteeing that the specifications,safety and efficacy of its analgesic and anti-inflammatory propertiesremain reproducible and consistent.

In the light of the above, a process for obtaining a standardizedextract from Aleurites moluccana, a process for isolating its marker anda process for preparing a phytotherapeutic pharmaceutical compositionhaving analgesic, anti-inflammatory and antipyretic properties as wellas a treatment method and its use as an alternative therapy to NSAIDs inview of its therapeutic potential and, possibly, better safety andtolerability characteristics are all obviously particularly interesting.

SUMMARY OF THE INVENTION

The present invention refers to a process for obtaining a standardizedextract having antinociceptive, anti-inflammatory and antipyreticproperties, from a plant of genus Aleurites, preferentially, Aleuritesmoluccana L. Willd, using, at least, one part of the plant,preferentially the leaves, with the said standardized extract beingdistinguished by the marker 2″-O-rhamnosylswertisin. The inventionfurther provides a pharmaceutical composition comprising the saidstandardized extract, methods of treatment for prevention or control ofpainful, inflammatory or febrile affections and uses of the saidextract.

The invention also refers to the pharmacological characterisation of theabove mentioned standardized extracts, with reference to theirantinociceptive, anti-inflammatory, antipyretic, antiedematogenic,anti-hypernociceptive activities and their acute toxicity evaluation.

The invention further provides a pharmaceutical composition comprisingan active ingredient of as therapeutically efficient amount of astandardized extract of Aleurites moluccana containing at least oneactive ingredient selected from the group comprising of alpha-amyrin,beta-amyrin, alpha-amirinone, beta-amirinone or swertisin, with the saidextract being standardized in relation to its marker2″-O-rhamnosylswertisin. Preferentially, the pharmaceutical compositionof the invention comprising the standardized extract of Aleuritesmoluccana contains a 2″-O-rhamnosylswertisin rate in the range between0.05 to 150.

Finally, as an additional embodiment, the present invention comprises atreatment method and use of said extract, isolated or within apharmaceutical composition, to prevent, control ou treat conditions ofpain, inflammation and/or fever.

BRIEF DESCRIPTION OF THE DRAWINGS

The following figures are part of the present report and are includedhere so as to illustrate determined aspects of the invention. The objectof the present invention may be better understood referring to one ormore of these figures, combined with the detailed description of thepreferred mode presented herein.

FIG. 1 shows a chromatography profile of the results obtained from thesoft extract and dry extract of Aleurites moluccana, using a mixture ofAcOEt:Acetone:H₂O 25:8:2 as an eluant in (A) and using a mixture ofhexane:AcOEt 85:15 as an eluant in (B).

FIG. 2 shows the results obtained from the analysis of the extract ofAleurites moluccana by High Performance Liquid Chromatography (HPLC)with (A) being the dry extract and (B) being the soft extract.

FIG. 3 shows the results obtained from the analysis of the marker2″-O-rhamnosylswertisin by Carbon-13 Nuclear Magnetic Resonance (NMRC¹³) (300 MHz) in deuterated methanol (MeOD), with (A) being in theregion of 65 to 0 ppm (300 MHz); (B) being in the region of 86 to 54 ppm(300 MHz) and (C) being in the region of 205 to 85 ppm (300 MHz).

FIG. 4 shows the results obtained from the analysis of the marker2″-O-rhamnosylswertisin by Hydrogen-1 Nuclear Magnetic Resonance (NMRH¹) (300 MHz) in deuterated methanol (MeOD), with (A) being in theregion of 13 to 0 ppm (300 MHz); (B) being in the region of 8.5 to 5.5ppm (300 MHz) and (C) being in the region of 4.5 to 0.5 ppm (300 MHz).

FIG. 5 shows the HPLC chromatogram of the standard2″-O-rhamnosylswertisin purified by PTLC (AMSR170707) at 145 g/ml, with(A) being at 338 nm, and (B) being in the ultraviolet region at 213.8,270.3 and 336.8 nm.

FIG. 6 shows the results obtained for the marker 2″-O-rhamnosylswertisinwhen assessed by Infrared Spectroscopy with a KBr pellet.

FIG. 7 shows the results obtained from the extract of Aleuritesmoluccana, with (A) being the dry extract, and (B) being the softextract.

FIG. 8 shows the results obtained from the dry extract of Aleuritesmoluccana, with (A) being in Phase I, (B) being in Phase II and (C)being the Edema (control group).

FIG. 9 shows the results obtained from the soft extract of Aleuritesmoluccana, with (A) being in Phase I, (B) being in Phase II and (C)being the Edema (control group).

FIG. 10 shows the results obtained from the extracts of Aleuritesmoluccana, with (A) being with the dry extract (B) being the softextract.

FIG. 11 shows the effects of the dry extract of Aleurites moluccana (125to 500 mg/kg) and of dexamethazone, administered orally, with the pawedema model, being induced in (A) by carragenin, in (B) by bradykininand in (C) by histamine.

FIG. 12 shows the results obtained from the dry extract of Aleuritesmoluccana (125 to 500 mg/kg) and with Indomethacin (10 mg/kg),administered orally, with the carragenine induced pleurisy model inmice, using total leukocytes in (A); with exudation in (B); neutrophilsin (C); and mononuclear cells in (D).

FIG. 13 shows the results obtained from the dry extract of Aleuritesmoluccana (125 to 500 mg/kg) and with Indomethacin (10 mg/kg),administered orally, with the substance P induced pleurisy model inmice, using total leukocytes in (A); with exudation in (B); neutrophilsin (C); and mononuclear cells in (D).

FIG. 14 shows the results obtained from the dry extract of Aleuritesmoluccana (125 to 500 mg/kg) and with Indomethacin (10 mg/kg),administered orally, with the bradycinine induced pleurisy model inmice, using total leukocytes in (A); with exudation in (B); neutrophilsin (C); and mononuclear cells in (D).

FIG. 15 shows the results obtained from the dry extract of Aleuritesmoluccana (125 to 500 mg/kg) and with Indomethacin (10 mg/kg),administered orally, with the histamine induced pleurisy model in mice,using total leukocytes in (A); with exudation in (B); neutrophils in(C); and mononuclear cells in (D).

FIG. 16 shows the results obtained from the dry extract of Aleuritesmoluccana (125 to 500 mg/kg) and with Indomethacin (10 mg/kg),administered orally, with the croton oil-induced ear edema model.

FIG. 17 shows the results obtained from the dry extract of Aleuritesmoluccana (5 to 10 mg/kg) and Dipyrone (10 mg/kg), administered orally,with the LPS induced hyperthermia model.

FIG. 18 shows the results obtained from the soft extract of Aleuritesmoluccana (5 to 10 mg/kg) and Indomethacin (10 mg/kg), administeredorally, for verification of occurrence of a lesion to the gastricmucosa.

FIG. 19 shows the anti-hypernociceptive effects of the dry extract ofAleurites moluccana (125 to 500 mg/kg, administered orally) in themechanical hypernociception model induced by the intraplantar injectionof carragenine (300 μg/paw).

FIG. 20 shows the anti-hypernociceptive effects of the dry extract ofAleurites moluccana (125 to 500 mg/kg, administered orally) for themechanical hypernociception model induced by the intraplantar injectionof PGE2 (0.1 nmol/paw).

FIG. 21 shows the effect of preventive treatment using the dry extractof Aleurites moluccana (125 to 500 mg/kg, administered orally) formechanical hypernociception induced by the intraplantar injection of CFA(20 μl/paw).

FIG. 22 shows the effect of curative treatment using the dry extract ofAleurites moluccana (125 to 500 mg/kg, administered orally) formechanical hypernociception induced by the intraplantar injection of CFA(20 μl/paw) in the paw ipsilateral in (A) and contralateral in (B) tothe injection.

FIG. 23 shows the results obtained with a fraction of dichloromethane ofAleurites moluccana assessed by Gas Chromatography (GC).

FIG. 24 shows the effect of treatment using the dry extract of Aleuritesmoluccana (125 to 500 mg/kg, administered orally) for mechanicalhypernociception induced by PSNC.

DETAILED DESCRIPTION OF THE INVENTION

To facilitate full comprehension of the present invention, it shall beexplained in greater detail below by identifying its particularities andpreferred modes. However, the present description shall not be taken aslimiting the embodiments of the invention.

Processes for Obtaining Standardized Extracts

A process for obtaining a standardized extract is particularly useful tothe development of a phytomedicine by guaranteeing its quality, itsphytochemical constitution by the use of markers, its physicochemicalcharacteristics, its pharmacological properties and, consequently, itstherapeutic action in a reproducible manner.

Overall, the process for obtaining a standardized extract, from at leastone part of a plant of the genus Aleurites, having antinociceptive,anti-inflammatory and antipyretic properties, in accordance with thepresent invention, comprises the following stages:

collection, drying and subdivision of the plant material;

pre-extraction; exraction with an extractor solvent;

filtration of the extract;

concentration of the extract

pasteurisation;

drying; and

qualitative and quantitative analysis (standardisation) of the extract.

The stages of the process, in accordance with the present invention, aresufficient to ensure an adequate concentration of, at least, one of thefollowing compounds: alpha-amyrin, beta-amyrin, alpha-amirinon,beta-amirinon, swertisin and 2″-O-rhamnosylswertisin. More specifically,the stages of the process are capable of providing the standardizedextract with a total concentration of flavonoids attaining between 0.1to 15% of its constitution. More preferentially, the stages of theprocess are capable to provide a standardized extract with a totalconcentration of 2″-O-rhamnosylswertisin attaining between 0.05 to 15%of its constitution.

In accordance with the present invention, the collection, drying andsubdivision stage is performed with the plant material obtained from apart of a plant from the genus Aleurites which include: A. trisperma, A.cordata, A. montana, A. fordii, A. montance, A. rockinghamensis and A.moluccana. Preferentially, the plant material used is from a part of thespecies Aleurites moluccana L. Willd, and more specifically, from itsleaves.

The plant material can be obtained advantageously from healthyindividuals of the species A. moluccana, whether adult (10 years), young(<10 years), intact or in shoot stage, and may be collected in any monthof the year, any season and any hour or period of the day. However, thecollection of the plant material is especially useful when undertaken inwinter due to the better phytochemical profile of the species in thisperiod.

In accordance with the present invention, the plant material may be usedfresh, dried, whole, torn, chopped, shredded, ground, powdered ormicronised. More specifically, the use of the plant material dried andshredded in particles with a size of 50 mm or less.

The process for obtaining the standard extract in accordance with thepresent invention requires a pre-extraction stage corresponding towashing the plant material with an adequate solvent, preferentiallyethanol. The pre-extraction stage is especially advantageous foreliminating the contaminants or impurities adsorbed by the plantmaterial, particularly heavy metals (eg. lead and mercury) adsorbed bythe leaf, which may contaminate the final standard extract unless theyare eliminated. In a preferred embodiment of the pre-extraction stage,the plant material is washed with a sufficient quantity of ethanol 96°GL, under constant agitation, at room temperature, for 15 to 30 minutes,with the solvent being drained and eliminated after this period.

The extraction stage of the process using extractor solvent inaccordance with the present invention comprises an extraction proceduresuch as, but not limited to, maceration, percolation, decoction,infusion, lixiviation, distillation, turbo extraction, supercriticalextraction or similar, using an adequate extractor solvent.

Examples of extractor solvents include, but are not limited to water,methanol, ethanol, propanol, isopropanol, propylenoglycol, acidsolution, ethyl acetate, dichloromethane, chloroform, hexane, glycerine,acetone, petroleum ether, supercritical fluid, and a combination thereofand similar.

Preferentially, in accordance with the present invention, the extractionprocess consists in maceration and the extractor solvent is ahydro-alcoholic solution of water:ethanol, in which the ratio of wateris less than the ratio of alcohol, i.e. using ratios of 1:9, 2:8, 3:7,4:6 and 1:1. But, preferentially, the water:alcohol ratio is of 3:7. Theextraction may occur in the presence or absence of light and/or oxygen,under agitation or not, for a period of approximately ≦10 days.

The extraction may occur in the presence or absence of light and/oroxygen, under agitation or not, for a period of approximately ≦10 days.

The ratio of extractor solvent:plant material used in the extractionprocedure may vary from 1:1 to 20:1. More specifically, the extractorsolvent:plant material is of 10:1.

In accordance with the present invention, the process for obtaining theextracts requires a filtration process for the elimination of the plantmaterial residue. The filtration procedures include, but are not limitedto deep or superficial filtration, using different types of filtermaterials.

The extract concentration process stage may be undertaken through theuse of a procedure selected amongst techniques of reduced-pressureevaporation, heat-convection evaporation, heating, a combination thereofand similars.

More preferentially, the extract concentration procedure refers to thereduced-pressure evaporation technique, using a heating temperature ofapproximately ≦70° C. and a vacuum between 200 and 400 mmHg, which iscapable of providing a concentration of approximately 20% to 50% solidsin the extract and sufficient to eliminate 100% of the alcohol.

Following concentration of the extract, the pasteurisation stage of thesoft extract aiming to extinguish eventual microbial contamination isespecially useful to the process. The pasteurisation stage isparticularly applicable when performed at a temperature of approximately95° C. for 3 minutes, at least.

It is known that plant extracts generally contain pigments, such aschlorophyll and insoluble materials that may lead to an undesirablevisual aspect of the extracts. Therefore, in accordance to a preferredaspect of the present invention, a stage for the elimination of theseelements or depigmentation may optionally be used following the extractconcentration stage in the process for obtaining a standard extract.Pigments and insoluble materials may be partly or totally removed fromthe extracts, for example, through treatment with active charcoal oradsorption resin, membrane ultrafiltration and the similar.

More specifically, the use of ultrafiltration through polymericmembranes with pore sizes between 5 and 100 kDa, under the impulse ofgravity or pressure.

The membrane ultrafiltration may also be useful for removing oxidasesfrom extracts, especially those with a high molecular weight, such aslaccase. The elimination of oxidases may be especially desirable for thestabilisation of standard extracts.

Other processes are known in the state of the art for performing theextract stabilization stage. It is possible, for example, to stabilisean extract through the addition of a sufficient quantity of one or morestabilisation agents or antioxidants such as, but not limited to:butylhydroxytoluene, butylhydroxyanisol, ascorbic acid and itsderivatives, octyldodecanol, cysteine, glutathione, and similars. Thus,optionally, it is possible to include an extract stabilisation stage,relating to the addition of a stabilising agent at a concentration of0.01 to 5% and preferably of 0.2 to 1% based on the extract. Inaccordance with a preferred aspect, a stabilising agent may be addedduring or following the extraction process.

In accordance with an alternative mode of the present invention, adrying stage of the extract after the concentration stage may be usedfor obtaining a standard dry extract.

The drying procedure, in accordance with the present invention may beselected from a group comprising lyophilisation, drying by atomisation(spray-drying), evaporation under reduced pressure, drying by heatconvection, or a combination thereof. More preferentially, the dryingprocedure in accordance with the present invention refers to thespray-drying technique.

Drying adjuvants may be used advantageously in the drying procedure byspray-drying. Examples of drying adjuvants include, but are not limitedto: colloidal silicon dioxide, modified cassaya starch, tricalciumphosphate, maltodextrin, cyclodextrins, microcrystalline cellulose,lactose, a combination thereof and similars. The use of drying adjuvantsis especially useful in quantities of 10 to 40% in relation to the ratioof total solids in the final product.

In accordance with an optional aspect, an additional microbialdecontamination stage may be performed after the drying stage. Thetechniques of gamma ray radiation, ultraviolet radiation and similarsare particularly useful in this case.

The process for obtaining a standard extract in accordance with thepresent invention requires a qualitative and quantitative analysis stageby means of duly validated analytic methodologies associated tolinearity, accuracy, precision, specificity and robustness.

In accordance with the present invention, the analytic methodologiesparticularly useful for the qualitative and quantitativecharacterisation include, but are not limited to Thin layerchromatography (TLC), paper chromatography, Gas Chromatography (GC),column chromatography (CC), high performance liquid chromatography(HPLC), mass spectrometry (MS), thermo-analytic techniques and thesimilar.

In accordance with the present invention, a standardized extract may bemore particularly analysed by TLC, presenting a chromatographic profilethat is peculiar such as that presented in FIGS. 1 (A and B) and by HPLCas presented in FIGS. 2 (A and B). FIG. 1 (A) shows the results of thechromatogaphic profile obtained for the soft extract and the dry extractof Aleurites moluccana, when assessed on a chromatographic plate,developed using a mixture of AcOEt:Acetone:H₂O 25:8:2 as an eluant andrevealed with a solution of ferric chloride 3%. The first stain refersto the soft extract (EtOH:H₂O 9:1), the second stain to the dry extract,the third stain to the swertisin standard and the fourth to2″-O-rhamnosylswertisin. FIG. 1 (B) shows the results of thechromatogaphic profile obtained for the soft extract and the dry extractof Aleurites moluccana, when assessed on a chromatographic plate,developed using a mixture of hexane:AcOEt 85:15 and revealed with asolution of hot sulphuric anisaldehyde. The first stain refers to thesoft extract (EtOH:H₂O 9:1), the second stain to the dry extract, thethird stain to the alpha and beta-amyrin standard, the fourth to alphaand beta-amirinon standard and the fifth stigmasterol andbeta-sitosterol standard. FIG. 2 (A) shows the results obtained for thedry extract of Aleurites moluccana when assessed by High performanceliquid chromatography (HPLC). The extract was initially dissolved inmethanol (20% of final volume), sonicated for 5 min, followed by theaddition of water acidified to pH 3.57 (20% of final volume). The volumewas then completed with a mixture of methanol/water acidified 50:50 to aconcentration of 1 to 2 mg/ml. After filtration of the sample through a0.22 m membrane filter, 20 L of the solution was injected by means of areversed phase column, where groups C₁₈ are chemically linked to silica,presenting a length of 10 to 25 cm and an internal diameter of 4 to 6mm. A PDA (diode array) type detector was used to monitor thechromatographic run at 254 and 338 nm. Separation was performed using alinear gradient mobile phase, composed initially of methanol/wateracidified acidified to pH 3.57: acetonitrile (20:15:65) at a flow of 0.5to 1 ml/min, to a proportion of 10:15:75, after 20 min, with a pressurevariation of 2.000 to 3.000 psi. FIG. 2 (B) shows the results obtainedfor the soft extract of Aleurites moluccana when assessed by Highperformance liquid chromatography (HPLC) in the same manner used forFIG. 2 (A).

In accordance with the present invention, a standard dry extract or softextract is particularly characterised by presenting a2″-O-rhamnosylswertisin marker ratio of 0.05 to 15% of its composition.

In accordance with the present invention, a fractioning stage of thestandard extract may be particularly useful for concentrating thesubstances potentially related to pharmacological activities of thespecies. Several fractioning processes are known in the state of theart. More particularly, the use of a column with differentchromatographic supports (i.e. silica, Sephadex®, aluminum, celluloseand similars) and adequate eluents or liquid-liquid partition usinghexane, dichloromethane, chloroform, ethyl acetate, butanol and similarsis useful. More preferentially, hexane, dichloromethane, and chloroformmay be used to obtain a concentrated fraction relating to the substancesof non-polar character in the extract, such as beta-amyrin,alpha-amyrin; and ethyl acetate and butanol may be used to obtain aconcentrated fraction relating to the substances of polar character inthe extract, such as swertisin and 2″-O-rhamnosylswertisin.

The preferred embodiments of the present invention for obtaining andanalysing a standard extract are described in detail in Examples I, IIand III.

Isolation, Purification and Characterisation of the Marker

The selection, isolation, purification and characterisation of aconstituent/marker of an extract are particularly necessary for thequantification and standardisation of the extracts.

In accordance with the present invention, the flavonoid2″-O-rhamnosylswertisin was established as being the most appropriatemarker for the process of obtaining a standard extract. This selectionis justified because it constitutes a main compound of the plant, it ismore discriminatory of the species, it is associated to thepharmacological properties of the extract and by the fact of being aflavonoid, which may be indicative of the extract's stability, duringtechnological processing or storage. (Sonaglio, D. et al.,Desenvolvimento Tecnológico e Produção de Fitoterápicos. In: SIMÕES etal. (Org.) Farmacognosia: Da Planta ao Medicamento. Porto Alegre:Editora da Universidade, 1999, 221-258).

These quantification and standardisation procedures of the extractsoccur according to the following stages:

I. Isolation,

II. Purification, and

III. Characterisation of the Marker.

The isolation of the 2″-O-rhamnosylswertisin marker (Stage I) may beperformed by techniques that include, but are not limited to,liquid-liquid partition, preparative TLC (PTLC), column chromatography,centrifuge chromatography, preparative or semi-preparative HPLC (PHPLC),a combination thereof and similars.

More preferentially, the isolation of the 2″-O-rhamnosylswertisin markermay be performed using, sequentially, the techniques of liquid-liquidpartition, open column chromatography and preparative TLC (PTLC), usingsilica 60 as stationary phase for the two latter and achloroform:methanol mixture as an eluant or preparative orsemi-preparative HPLC using preparative or semi-preparative C18 typereversed phase columns with a particle size of 10 μm, using a lineargradient of methanol:acetonitrile:acidified water as a mobile phase,monitored at 254 to 338 nm.

The purification of 2″-O-rhamnosylswertisin marker (Stage II) may beperformed by techniques that include, but are not limited to,preparative TLC and preparative or semi-preparative HPLC.

More preferentially, the purification of the 2″-O-rhamnosylswertisinmarker may be performed using preparative TLC, using silica 60 asstationary phase and a chloroform:methanol mixture as an eluant.Alternatively, the preparative or semi-preparative HPLC technique may beused, using preparative or semi-preparative C₁₈ type reversed phasecolumns with a particle size of 10 μm, using a linear gradient ofmethanol:acetonitrile:acidified water as a mobile phase, monitored at254 to 338 nm.

In accordance with the present invention, the use of the above mentionedprocedures should achieve a purity rate equivalent to approximately>90%.

In accordance with the present invention, the qualitative andquantitative analysis of the isolated and purified2″-O-rhamnosylswertisin marker, or in other words, its characterisation(Stage III) may be performed using techniques that include, but are notlimited to hydrogen and carbon-13 nuclear magnetic resonance (NMR),infrared spectrophotometry (IV), ultraviolet spectrophotometry (UV),HPLC, TLC and thermal analysis.

The isolated and purified 2″-O-rhamnosylswertisin marker is particularlycharacterised by NMR and HPLC. FIG. 3 shows the results obtained withthe marker when assessed by Carbon-13 Nuclear Magnetic Resonance (NMRC¹³), (300 MHz) in deuterated methanol (MeOD), with (A) being in theregion of 65 to 0 ppm (300 MHz); (B) being in the region of 86 to 54 ppm(300 MHz) and (C) being in the region of 205 to 85 ppm (300 MHz). FIG. 4shows the results obtained with the 2″-O-rhamnosylswertisin marker whenassessed by hydrogen Nuclear Magnetic Resonance (NMR H¹), (300 MHz) indeuterated methanol (MeOD), with (A) being in the region of 13 to 0 ppm(300 MHz); (B) being in the region of 8.5 to 5.5 ppm (300 MHz) and (C)being in the region of 4.5 to 0.5 ppm (300 MHz). The characterisation ofthe marker by HPLC is shown in FIG. 5 which shows the chromatographicprofile by means of a HPLC chromatogram of the standard2″-O-rhamnosylswertisin purified by PTLC (AMSR170707) at 145 g/ml, in338 nm, which eluted in approximately 17 min, thus demonstrating successin purification of the marker, as indicated by the presence of a singlepeak on the chromatogram. FIG. 5 (B) shows the results and morespecifically the absorption profile of a typical flavonoid peak,obtained with 2″-O-rhamnosylswertisin marker with maximum absorptions of213.8, 270.3 and 336.8 nm when assessed by scanning the ultravioletspectrum in the Photo Diode Array (PDA) of the Liquid Chromatograph, inthe same manner as for FIG. 2 (A). FIG. 6 shows the infra-red absorptionspectrum typical of flavonoids.

A preferred embodiment of the present invention for the isolation,purification and analysis of the marker is described in more detail inExample IV.

Pharmacological Characterisation of the Standard Extracts

The standard extracts, in accordance with the present invention, may becharacterised as to their pharmacological activity in animal pain modelsby nociception, hypernociception, neuropathic pain, inflammation andfever.

Several animal models may be applied to the pharmacologicalcharacterisation of an extract with antinociceptive, anti-inflammatoryand antipyretic properties, and these include: 1. Abdominal contortiontests induced by acetic acid; 2. Randal-Sellito Test; 3. Mechanicalhypernociception models (of inflammatory and/or neuropathic origin); 4.Formalin test; 5. Hot plate assays; 6. Neuropathic pain assays; 7. Pawand ear edema trials in rats and/or mice, induced by differentphlogistic agents; 8. Pleurisy model, induced by different phlogisticagents; 9. Arthritis models induced by Freund adjuvants; 10.Hyperthermia models induced by LPS; 11. Evaluation of undesirableeffects; and similars.

The standard extracts, in accordance with the present invention, whencharacterised as to their pharmacological activity, demonstrateantinociceptive, anti-inflammatory and antipyretic properties, asdescribed in greater detail below.

Antinociceptive Effects of the Extracts of Aleurites moluccana Using theAbdominal Contortion Model Induced by Acetic Acid.

The oral administration of the dry and soft extracts is capable ofsignificantly inhibiting in a dose dependent manner the number ofcontortions induced by intraplantar injections of acetic acid in mice,and DI50s of 62.65 (24.99-157.64) mg/kg, 99.84 (85.30-116.85) mg/Kg,respectively. FIG. 7 shows the results obtained with the extracts ofAleurites moluccana, in (A) (dry extract) and in (B) (soft extract),administered orally, in the abdominal contortion model induced by aceticacid. Each column represents the average of 8 to 10 animals and thevertical bars indicate the ASEs. The results differ significantly inrelation to the control group, **P<0.01. The inhibition values (IM)obtained are stipulated at 68.64±1.22%; 55.12±2.13% and 64.0±1.65%;63.5%, and 96.9%, for the dry and soft extracts, respectively, as shownin Table 1 below, compared to the pharmaceuticals used in clinicalpractice.

TABLE 1 Comparative data of DI50 and IM in the abdominal contortionmodel induced by acetic acid: oral administration. Group IM (%) DI50(mg/kg) Dry Extract 68.6 62.65 (24.99-157.08) Soft extract 55.12 99.84(85.30-116.85) Indomethacin 72.0 189.95 (142.51-253.17) AAS 64.5 364.1(303.4-437.0)  Diclophenac 51.6 443.07 (396.18-495.50)

Antinociceptive Effects of the Extracts of Aleurites moluccana Using thePain Model Induced by Formalin.

The oral administration of the dry extract, in doses of 50 to 500 mg/kg,is capable of significantly inhibiting in a dose dependent manner boththe neurogenic phase and the inflammatory phase of nociception inducedby intraplantar injections of formalin, with an inhibition of 69±2.34%and 15.84±1.98% for the first and second phase of formalin,respectively, and DI50% of 271.83 (248.84-296.94) mg/kg for the firstphase of the test. FIG. 8 shows the results of this experiment, withPhase I being in (A), Phase II being in (B) and the Edema being in (C).Each column represents the average of 8 to 10 animals and the verticalbars indicate the ASEs. The results differ significantly in relation tothe control group (C), *P<0.05 and **P<0.01 or, in other words, the sameextract was not capable of reducing the edema of the paw induced byformalin (FIG. 8 (C)).

The oral administration of the soft extract of Aleurites moluccana, indoses of 50 to 500 mg/kg administered orally, is capable ofsignificantly inhibiting in a dose dependent manner both the neurogenicphase and the inflammatory phase of nociception induced by intraplantarinjections of formalin, with an inhibition of 46.5±3.41% and 79.08±2.54%for the first and second phase of formalin, respectively, and DI50%superior to 500 mg/kg for the second phase of the test. FIG. 9 shows theresults of this experiment in the formalin induced pain model, withPhase I being in (A), Phase II being in (B) and the Edema being in (C).Each column represents an average of 8 to 10 animals and the verticalbars indicate the ASEs. The results differ significantly in relation tothe control group (C), *P<0.05 and **P<0.01. The same extract wascapable of reducing the edema of the paw induced by formalin with aninhibition of 34% (FIG. 9 (C)). The IM and DI50 values compared to thepharmaceuticals used in clinical practice are shown in Table 2 (Phase I)and Table 3 (Phase II) below.

TABLE 2 Comparative data of DI50 and IM - formalin induced pain model -Phase I: oral administration. Group IM (%) DI50 (mg/kg) Dry Extract 69.0271.83 (248.84-296.94) Soft extract 46.5 >500 Indomethacin 7.2 >300 AAS12.7 >500 Diclophenac 68.8 407.48 (357.56-464.37)

TABLE 3 Comparative data of DI50 and IM - formalin induced pain model -Phase II: oral administration. Group IM (%) DI50 (mg/kg) Dry Extract15.8 >500 Soft extract 79.1 ~150 Indomethacin 53.1 263.42(239.83-289.35) AAS 65.7 408.87 (391.47-427.03) Diclophenac 82.5 279.69(245.47-318.67)

Table 4 below shows the IM and DI50 values—formalin induced pain modelfor evaluation of the Edema compared to the pharmaceuticals used inclinical practice.

TABLE 4 Comparative data of DI50 and IM - formalin induced pain model -Edema: oral administration. Group IM (%) DI50 (mg/kg) Dry Extract17.4 >500 Soft extract 34.0 >500 Indomethacin 36.5 >300 AAS 31.7 >500Diclophenac 33.7 >500

Antinociceptive Effects of the Extracts of Aleurites moluccana Using theHot Plate Model.

FIG. 10 represents the results obtained with the dry (A) and soft (B)extracts of Aleurites moluccana, administered orally, with the hot platemodel. Each point represents the average of 8 to 10 animals and thevertical bars indicate the ASEs. The results differ significantly inrelation to the control group (C), **P<0.01. None of the extractsadministered orally were capable of significantly altering the latencyof the animals in the hot plate test compared to morphine, thepharmaceutical of choice for the positive control, as can be seen inFIG. 10.

Anti-Inflammatory Effects of the Extracts of Aleurites moluccana usingthe paw edema model induced by Phlogistic Agents.

As shown by FIGS. 11 (A and B), the oral administration of the dryextract of Aleurites moluccana (125 to 500 mg/kg) is capable ofsignificantly reducing paw edema induced by carragenine and bradycinin.However, the same extract is only capable of significantly reducing pawedema induced by histamine at one point (30 min) (FIG. 11 C).

Anti-Inflammatory Effects of the Dried Extract of Aleurites moluccanaUsing the Pleurisy Model Induced by the Intrapleural Injection ofPhlogistic Agents.

FIG. 12 represents the results obtained with the dry extract ofAleurites moluccana (125 a 500 mg/kg), and with Indomethacin (10 mg/kg),administered orally, in the pleurisy induced by carragenine model inmice. Each point represents an average of 8 to 10 animals and thevertical bars indicate the ASEs. The results differ significantly inrelation to the control group (C), *P<0.05 and **P<0.01. # representsthe difference between the control group and the saline solution group.The total leukocytes are in (A); with exudation being in (B);neutrophils being in (C); and mononuclear cells being in (D). As can beseen in FIG. 12, the pre-treatment with the dry extract of Aleuritesmoluccana, administered orally, in doses of 125-500 mg/kg, is capable ofinhibiting exudation as well as the migration of inflammatory cells,represented by the count of total leukocytes, mononuclear cells andneutrophils, in the pleurisy induced by carragenine model in mice.However, the same is only capable of acting on the plasmatic outflow andon the migration of mononuclear cells in the pleurisy induced bysubstance P model (see FIGS. 13 A to D). FIG. 13 shows the resultsobtained with the dry extract of Aleurites moluccana (125 a 500 mg/kg),and with Indomethacin (10 mg/kg), administered orally, in the pleurisyinduced by substance P model in mice. Each point represents an averageof 8 to 10 animals and the vertical bars indicate the ASEs. The resultsdiffer significantly in relation to the control group (C), *P<0.05 and**P<0.01. # represents the difference between the control group and thesaline solution group. The experiment was conducted with totalleukocytes (FIG. 13(A)); by exudation (FIG. 13 (B)); with neutrophils(FIG. 13(C)); and with mononuclear cells (FIG. 13(D)).

FIG. 14 represents the results obtained with the dry extract ofAleurites moluccana (125 a 500 mg/kg), and with Indomethacin (10 mg/kg),administered orally, in the pleurisy induced by bradykynin model inmice. Each point represents an average of 8 to 10 animals and thevertical bars indicate the ASEs. The results differ significantly inrelation to the control group (C), *P<0.05 and **P<0.01. # representsthe difference between the control group and the saline solution group.The total leukocytes are in (A); with exudation being in (B);neutrophils being in (C); and mononuclear cells being in (D). As can beseen in FIG. 14, the pre-treatment with the dry extract of Aleuritesmoluccana, administered orally, in doses of 125-500 mg/kg, is capable ofinhibiting exudation as well as the migration of inflammatory cells,represented by the count of total leukocytes, mononuclear cells andneutrophils in the pleurisy induced by bradykynin model in mice.However, the same is only capable of acting on the plasmatic outflow (ina dose dependant manner) and the migration of mononuclear cells in thepleurisy induced by histamine model (see FIGS. 13 B to D). FIG. 15 showsthe results obtained with the dry extract of Aleurites moluccana (125 a500 mg/kg), and with Indomethacin (10 mg/kg), administered orally, inthe pleurisy induced by histamine model in mice. Each point representsan average of 8 to 10 animals and the vertical bars indicate the ASEs.The results differ significantly in relation to the control group (C),*P<0.05 and **P<0.01. # represents the difference between the controlgroup and the saline solution group. The experiment was conducted withtotal leukocytes (FIG. 15(A)); by exudation (FIG. 15 (B)); withneutrophils (FIG. 15(C)); and with mononuclear cells (FIG. 15(D)).

Antiedematogenic Effects of the Dried Extract of Aleurites moluccanaUsing the Edema of the Ear Model Induced by Croton Oil.

FIG. 16 represents the results obtained with the dry extract ofAleurites moluccana (125 a 500 mg/kg), and with Indomethacin (10 mg/kg),administered orally, in the edema of the ear induced by croton oilmodel. Each point represents an average of 8 to 10 animals and thevertical bars indicate the ASEs. The results differ significantly inrelation to the control group (black bar), *P<0.05 and **P<0.01. FIG. 16demonstrates that the oral administration of the dry extract isefficient in reducing the edema of the ear induced by croton oil with anIM calculated at 43.75±2.47%.

Antipyretic Effects of the Extracts of Aleurites moluccana Using theHyperthermia Model Induced by LPS in Rats.

FIG. 17 shows the results obtained with the dry extract of Aleuritesmoluccana (5 a 10 mg/kg), and Dipyrone (10 mg/kg), administered orally,in the hyperthermia induced by LPS model. Each point represents anaverage of 8 to 10 animals and the vertical bars indicate the ASEs. Theresults differ significantly in relation to the control group, *P<0.05.# represents the difference between the control group and the salinesolution group. FIG. 17 demonstrates that the oral administration of thedry extract promotes a reduction of the febrile response in the animalsin a statistically significant manner when compared to the controls(Dipyrone, vehicle) and does not produce alterations to the gastricmucosa, normally present in antipyretic agents.

Effects of the Extracts of Aleurites moluccana in Inducing Lesions tothe Gastric Mucosa in Rats.

FIG. 18 shows the results obtained with the soft extract of Aleuritesmoluccana (5 a 10 mg/kg), and Indomethacin (10 mg/kg), administeredorally, for verification of lesions to the gastric mucosa. Each pointrepresents an average of 8 to 10 animals and the vertical bars indicatethe ASEs. The results differ significantly in relation to the controlgroup, *P<0.05. # represents the difference between the control groupand the saline solution group. FIG. 18 demonstrates that the oraladministration of the soft extract does not produce alterations to thegastric mucosa, normally present in antipyretic agents.

Effects of the Dry Extract of Aleurites moluccana on MechanicalHypernociception Induced by the Intraplantar Injection of Carragenin.

FIG. 19 shows the anti-hypernociceptive effect of the dry extract ofAleurites moluccana (125 a 500 mg/kg, v.o.) using the mechanicalhypernociception induced by the intraplantar injection of carrageninemodel (300 mg/paw). Each point represents the average of 6 to 8 animalsand the vertical bars indicate the ASEs. The results differsignificantly in relation to the control group, *P<0.05, **P<0.01e***<0.001. As can be seen in FIG. 19, the oral treatment with the dryextract of Aleurites moluccana, when administered in doses of 125, 250and 500 mg/kg, is capable of significantly reducing in a dose dependentmanner the mechanical hypernociception induced by the injection ofcarragenin, with a DI50% of 433 (295 to 407) mg/kg. This inhibition isobserved for up to 6 hours after the injection with carragenine. Theinhibition observed in relation to the control response are stipulatedat 33±5% and 51±2%, for the doses of 250 and 500 mg/kg, respectively.

Effects of the Dry Extract of Aleurites moluccana on MechanicalHypernociception Induced by the Intraplantar Injection of ProstaglandinE₂ (PGE₂).

FIG. 20 shows the anti-hypernociceptive effect of the dry extract ofAleurites moluccana (125 a 500 mg/kg, v.o.) using the mechanicalhypernociception induced by the intraplantar injection of PGE2 model(0.1 nmol/paw). Each point represents the average of 6 to 8 animals andthe vertical bars indicate the ASEs. The results differ significantly inrelation to the control group, *P<0.05, **P<0.01 e***<0.001. FIG. 20demonstrates that the oral treatment with the dry extract of Aleuritesmoluccana, (125, 250 and 500 mg/kg) is capable of significantly reducingin a dose dependent manner the mechanical hypernociception responseinduced by the intraplantar injection of PGE₂, (for up to 2 hours afterthe injection of PGE₂), with DI50% equal to 201 (91 to 445) mg/kg.Inhibitions of 8±6%, 11±8% and 63±7% were obtained for the doses of 125,250 and 500 mg/kg, respectively.

Effects of the Dry Extract of Aleurites moluccana on MechanicalHypernociception Induced by the Intraplantar Injection of the CompleteFreund's Adjuvant (CFA).

FIG. 21 shows the effect of the preventive treatment with the dryextract of Aleurites moluccana (125 a 500 mg/kg, v.o.) on theanti-hypernociceptive effect of using the mechanical hypernociceptioninduced by the intraplantar injection of CFA model (20 μl/paw). Eachpoint represents the average of 6 to 8 animals and the vertical barsindicate the ASEs. The results differ significantly in relation to thecontrol group, *P<0.05, **P<0.01 e***<0.001. FIG. 21 demonstrates thatpreventive oral administration with the dry extract of Aleuritesmoluccana (125, 250 e 500 mg/kg) is capable of significantly reducingthe mechanical hypernociception induced by the intraplantar injection ofCFA, for up to 6 hours after the inducement of hypernociception, withD150% of 275 (239 to 316) mg/kg and inhibitions of 29±6% and 53±6% forthe doses of 250 and 500 mg/kg, respectively.

For assessment of the effect of the dry extract of Aleurites moluccanaon an existing condition of mechanical hypernociception, the animalswere first administered an intraplantar injection of CFA, and after 24 hwere treated with the extract in doses of 125, 250 and 500 mg/kg, 2times a day during 5 consecutive days.

FIG. 22 shows the effect of curative treatment using the dry extract ofAleurites moluccana (125 to 500 mg/kg, v.o.) on the mechanicalhypernociception induced by the intraplantar injection of CFA (20μl/paw) in the ipsilateral (FIG. 16(A)) and contralateral (FIG. 16(B))paw to the injection. Each point represents the average of 6 to 8animals and the vertical bars indicate the ASEs. The results differsignificantly in relation to the control group, *P<0.05, **P<0.01e***<0.001. FIG. 22 demonstrates that administration of the dry extractof Aleurites moluccana is capable of reverting an existing condition ofmechanical hypernociception (125, 250 e 500 mg/kg) is capable ofsignificantly reducing the mechanical hypernociception induced by theintraplantar injection of CFA, in the paw ipsilateral to the injectionin a dose dependant manner, with DI50% of 272 (130 to 568) mg/kg. Theinhibitions were of 8±6%, 11±8% e 63±7 for the doses of 125, 250 and 500mg/kg, respectively (FIG. 22 A). Similarly, the same treatment iscapable of preventing the occurrence of mechanical hypernociception inthe paw contralateral to the injected paw, with an inhibition of 56±8%in a dose of 500 mg/kg (FIG. 22 B).

A preferred embodiment of the tests for the pharmacologicalcharacterisation of the antinociceptive, anti-inflammatory andanti-hypernociceptive activities of the standard extracts of Aleuritesmoluccana obtained in accordance with the processes described above aredescribed in greater detail in the Examples V, VI and VII, below.

Toxicological Evaluation of the Soft Extract of Aleurites moluccana.

Preliminary studies with mice demonstrated that the oral administrationof the soft extract of Aleurites moluccana in a dose of 5000 mg/kg(n=10, 5 males and 5 females), did not cause any death in a period of 3to 24 hours. Furthermore, there were no signs of irritability,tachycardia, tremors or piloerection, as described in Tables 5 and 6.

TABLE 5 Evaluation of the behavioural parameters of male mice treatedwith the soft extract of Aleurites moluccana (5000 mg/Kg) using thehippocratic test. Evaluated Dose 0 to Parameters g/kg 10 min 30 min 60min 180 min 1.440 min Piloerection Control 1/5 1/5 0/5 0/5 0/5 Treated0/5 0/5 0/5 0/5 0/5 Cage corner Control 5/5 3/5 0/5 0/5 0/5 movementTreated 5/5 4/5 0/5 0/5 0/5 Tremors Control 0/5 0/5 0/5 0/5 0/5 Treated0/5 0/5 0/5 0/5 0/5 Tachycardia Control 0/5 0/5 0/5 0/5 0/5 Treated 1/50/5 0/5 0/5 0/5 Irritability Control 3/5 3/5 3/5 0/5 0/5 Treated 1/5 1/50/5 0/5 0/5 Miction Control 5/5 5/5 5/5 0/5 0/5 Treated 1/5 1/5 2/5 2/50/5 Defecation Treated 5/5 5/5 0/5 0/5 0/5 Control 4/5 2/5 0/5 0/5 0/5Ptosis Control 5/5 3/5 0/5 0/5 0/5 Treated 1/5 1/5 1/5 0/5 0/5Convulsion Control 0/0 0/0 0/0 0/0 0/0 Treated 0/0 0/0 0/0 0/0 0/0Deaths Control 0/0 0/0 0/0 0/0 0/0 Treated 0/0 0/0 0/0 0/0 0/0 PtosisControl 5/5 3/5 0/5 0/5 0/5 Treated 3/5 1/5 0/5 0/5 0/5 ConvulsionControl 0/0 0/0 0/0 0/0 0/0 Treated 0/0 0/0 0/0 0/0 0/0 Deaths Control0/0 0/0 0/0 0/0 0/0 Treated 0/0 0/0 0/0 0/0 0/0

TABLE 6 Evaluation of the behavioural parameters of female mice treatedwith the soft extract of Aleurites moluccana (5000 mg/Kg) using thehippocratic test. Evaluated Dose 0 to Parameters g/kg 10 min 30 min 60min 180 min 1.440 min Piloerection Control 3/5 2/5 0/5 0/5 0/5 Treated0/5 0/5 0/5 0/5 0/5 Cage corner Control 5/5 5/5 3/5 1/5 0/5 movementTreated 5/5 4/5 0/5 0/5 0/5 Tremors Control 3/5 3/5 0/5 0/5 0/5 Treated2/5 1/5 0/5 0/5 0/5 Tachycardia Control 0/5 0/5 0/5 0/5 0/5 Treated 0/50/5 0/5 0/5 0/5 Irritability Control 5/5 3/5 1/5 0/5 0/5 Treated 5/5 3/51/5 0/5 0/5 Miction Control 5/5 3/5 1/5 1/5 1/5 Treated 5/5 4/5 0/5 0/50/5 Defecation Control 5/5 3/5 2/5 1/5 0/5 Treated 5/5 4/5 1/5 0/5 0/5Deaths Control 0/0 0/0 0/0 0/0 0/0 Treated 0/0 0/0 0/0 0/0 0/0

A preferred embodiment of the tests for the pharmacologicalcharacterisation of the toxicological evaluation of the standardextracts of Aleurites moluccana obtained in accordance with theprocesses described above are described in greater detail in ExampleVIII, below.

Pharmaceutical Compositions

An extract of Aleurites moluccana obtained and characterised inaccordance with the above description may be administered singly, butshall generally be administered in combination with one ore morepharmaceutical vehicles or adjuvants, selected according to the route ofadministration and Galenic practices.

Pharmaceutical compositions having antinociceptive, anti-inflammatoryand antipyretic properties, for use in mammals, comprising an activeingredient of an efficient dose of at least one standard extract,obtained and characterised in accordance with the processes describedabove, in combination with one ore more pharmaceutical vehicles oradjuvants, also constitute aspects of the present invention.

More particularly, the object of the present invention is to provide apharmaceutical composition incorporating at least one extract ofAleurites moluccana comprising the compounds alpha-amyrin, beta-amyrin,alpha-amirinon, beta-amirinon, stigmasterol, beta-sitosterol,campesterol, swertisin and standardised in relation to its2″-O-rhamnosylswertisin marker having a ratio of 0.015 to 15%.

The pharmaceutical compound in accordance with the present invention maycontain a standard extract as defined above, in a quantity representingbetween 5 and 90% of the total weight of the composition, butpreferentially in a quantity representing between 20 and 80% of thetotal weight of the composition.

The pharmaceutical compound in accordance with the present invention maycontain a standard extract as defined above, in a concentration ofbetween 0.01 to 2000 mg per posological unit, but preferentially, in aconcentration of 50 to 1000 mg per posological unit.

The standard extracts and compositions of the present invention may beadministered orally, by means of oral pharmaceutical forms, such astablets, capsules (hard or soft), tablets, powders, granules, simplepills, coated pills, chewable pills, effervescent pills, sublingualpills, controlled release pills, dragees, globules, elixirs,suspensions, syrups and emulsions, each of which may includeformulatiuons for immediate, controlled, prolonged or delayed release.

The compositions containing the extract of the invention may also beadministered via topical route, by means of pharmaceutical forms, suchas ointments, unguents, creams, emulsions, gels, solutions, pastes,aerosols, transdermal systems and others known in the state of the art.

Intravenous (either bolus or infusion), subcutaneous or intramuscularadministration is also possible, by means of pharmaceutical forms knownin the state of the art.

Administration may also be intravaginal and rectal by means of thepharmaceutical forms: suppositories, ovules, ointments, creams andothers known in the state of the art.

In accordance with the present invention, the solid pharmaceuticalcomposition intended for oral administration should preferably be in theform of capsule or pill, containing at least one standard extract ofAleurites moluccana, which may be combined to one or morepharmaceutically acceptable inert vehicles, such as diluents,lubricants, anti-adherents, sliding agents, agglutinant agents,disaggregating agents, as well as conservatives, colourants, sweeteners,filmogen agents for coatings, plasticisers, opacifiers and the similar,as necessary. The quantities of these pharmaceutical vehicles oradjuvants may be equal to those conventionally used in the state of theart.

Examples of diluents include, but are not limited to starch, monohydratelactose, spray-dried lactose, microcrystalline cellulose, dicalciumphosphate, mannitol, powdered cellulose and the similar.

Examples of lubricants and sliding agents include, but are not limitedto: stearic acid, oxide of silicon, magnesium stearate, calciumstearate, talcum, stearile sodium fumarate, glyceryl triestearate,polyethylene glycol and the similar.

Examples of agglutinants include, but are not limited to pregelatinisedstarch, hydroxypropylcellulose, methylcellulose, sodiumcarboxymethylcellulose, hydroxypropylmethylcellulose,polyvinylpyrrolidone, starch, gelatine, natural sugars such as glucose,syrups, natural and synthetic gums, acacia, sodium alginate,polyethylene glycol, waxes and the similar.

Examples of desaggregating agents include, but are not limited tocarboxymethylcellulose, reticulated sodium, sodic carboxymethylcellulosewith a low substitution rate, sodic croscarmelose, sodium starchglycolate, cross linked polyvinylpyrrolidone and the similar.

Examples of sliding agents include, but are not limited to: colloidalsilicon dioxide, hydrophobic colloidal silicon dioxide and the similar.

Examples of conservatives include, but are not limited to ascorbic acid,peroxy benzoic acid esters, chlorobutanol, benzylic alcohol, phenethylalcohol, dehydroacetic alcohol and the similar.

Preferred examples of colorants include, but are not limited tohydro-unsoluble lacquer pigments, natural pigments, such as (3-caroteneand chlorophyll, iron oxide, yellow iron sesquioxide, black ironsesquioxide and the similar.

Examples of sweeteners include, but are not limited to: dextrose,mannitol, sorbitol, saccharose, glycerine, sodic saccharin, dipotassiumglycyrrhizinate, aspartame, stevia and the similar.

Examples of film coating polymers include, but are not limited tohydroxypropylmethylcellulose (e.g. Opadry®), hydroxypropylcellulose,cellulose acetophthalate and derivatives of methacrylic and methacrylateacid (e.g. Eudragit®) and the similar.

Examples of plasticisers and opacifiers include, but are not limited toglycerine fatty acid esters, triethyl citrate, propyleneglycol,polyethylene glycol, titanium dioxide and the similar.

The preferred embodiments of the present invention for obtaining a pilland a capsule are described below in greater detail in Example IX andExample X, respectively.

Pharmaceutical compositions intended for oral administration inaccordance with the present invention also include solutions, syrups,elixirs and suspensions that may contain, but are not limited tosolubilizers, solvents, co-solvents, dissolution adjuvants, humidifieragents, suspension agents, conservatives, stabilisers, alkalisers,acidifiers, viscosity modification agents, sweeteners and flavourisers.Overall, water, ethylic alcohol, appropriate oils, saline solution,aqueous dextrose (glucose), saccharose, glycerine, sugar relatedsolutions and glycols, such as propylenoglycol or polyethylene glycol s,phosphate buffer, cyclodextrins, agar, bentonite, carbomer,methylcellulose and hydroxypropylmethylcellulose are appropriatevehicles for liquid pharmaceutical forms.

The preferred embodiments of the present invention for obtaining asuspension or a syrup are described below in greater detail in ExampleXI and Example XII, respectively.

Pharmaceutical compositions in accordance with the present invention maybe pharmaceutical forms that propitiate modified, controlled, sustained,extended, prolonged, programmed, slow, delayed, enteric, colon-specificor site-specific release of the active ingredient. Examples ofpharmaceutical forms with these features include, but are not limited toosmotic pump systems, matricial systems, reservoir systems, ionicexchange systems, enteric coating forms, pH-dependant coating forms,transdermal systems, pulsing devices, implants, lipossomal systems,nano-structured systems, microstructures systems and the similar.

Pharmaceutical compositions in accordance with the present invention maybe obtained by industrial pharmaceutical processes and unitaryoperations known in the state of the art, such as, but not limited tomixing, grinding, sieving, shredding, micronisation, lubrication,classification, wetting, drying, dry granulation, humid granulation,compression, encapsulation, coating, dissolution and homogenisation, ora combination of these.

Preferentially, in accordance with the present invention, apharmaceutical composition intended for topical administration alsoinclude, but are not limited to emulsions, creams or ointments, maycontain one or more pharmaceutically acceptable inert vehicles oradjuvants. These adjuvants may be components of the aqueous phase,components of the oleous phase, emollients, humidifiers, emulsifiers,tensoactives, conservatives, stabilisers, antioxidants, chelatingagents, self-emulsifying waxes, bases for ointments, colorants, essencesand the similar, when necessary. The quantities of these pharmaceuticalvehicles or adjuvants may be equal to those commonly used in the stateof the art.

Examples of components of the oleous phase, consistency agents,emulsifiers and self-emulsifying waxes include, but are not limited tostearic acid, cetyl alcohol, stearyl alcohol, cetostearyl alcohol,cetostearyl alcohol 20oe (20 moles of ethylene oxide), glycerylmonostearate, propylenoglycol monostearate, sorbitan monostearate,microcrystalline wax, anionic self-emulsifying wax (for example,Lanete), non-ionic self-emulsifying wax (for example, Cosmowax®),non-ionic self-emulsifying wax (for example, Polawax®), non-ionicself-emulsifying wax (for example, Crodabase®) and the similar.

Examples of emollients include, but are not limited to isopropylmyristolate, mineral oil, myristyl alcohol, cetyl palmitate, octylpalmitate, myristyl myristate, glyceryl stearate, polyoxyethylenestearate, coconut's hydrogenated glycerids, lanolin, lanolin alcohols,decyl oleate, other esters and fatty acids and the similar.

Examples of components of the aqueous phase, consistency agents,humidifiers and polymers include, but are not limited to carbomer (forexample, Carbopol®), hydroxyethylcellulose (for example, Natrozol® andCellosize®), polyacrylic acid (for example, Pemulem®), propylenoglycol,sorbitol, glycerin, dimethicone, urea and the similar.

Examples of tensoactives include, but are not limited to tween 20, tween80, triethanolamin, polysorbate 80, octoxinol, sorbithane monopalmitate,sodium lauryl sulphate and the similar.

Examples of bases for ointments include, but are not limited to lanolin,vaseline, hydrophilic petrolate, yellow ointment, white ointment,hydrophilic ointment, polyethylene glycol ointment and the similar.

Examples of conservatives, stabilisers, antioxidants and chelatingagents include, but are not limited to methylparaben (for example,Nipagin®), propylparaben (for example, Nipazol®), butylparaben,timerosal, benzalconic chloride, benzetonium chloride, phenoxyethanol,phenoxyethanol+parabenes (for example, Phenonip®), diazolidinyl urea(for example, Germall®), diazolidinyl urea+iodopropynyl butylcarbamate(Germall Plus®), edetic acid, disodic EDTA, ascorbic acid, sodiumbisulphite, sodium metabisulphite and the similar.

With the purpose of complementing the therapeutic activity presentedherein, the pharmaceutical compositions including at least one extractin accordance with the present invention may combine, furthermore, otherpharmacologically active compounds such as synthetic and semi-syntheticsubstances, biological molecules (e.g. proteins) vitamins and otherderivatives of plant origin, depending on the intended effect.

Method of Treatment

Another aspect of the present invention refers to a method to prevent,control or treat clinical conditions of pain, inflammation and fever,with some of these having been presented above, that comprises theadministration, in mammals, of a therapeutically efficient dose of astandardized extract of Aleurites moluccana, or a pharmaceuticalcomposition that contains the latter, for an adequate period of time.

The method, in accordance with the present invention, is related to theclinical effects of an efficient dose of a standardized extract ofAleurites moluccana on illnesses and symptoms in which the substanceshaving antinociceptive, anti-inflammatory and anti-pyretic activity areparticularly useful.

The method, in accordance with the present invention, is particularlyuseful in the control of painful symptomologies of nociceptive origin,in acute, sub-acute and chronic conditions.

According to one embodiment of the present invention, the dose shouldcorrespond to between approximately 0.01 to 100 mg of extract perkilogramme of the patient, preferentially between approximately 0.1 to50 mg/kg, and more particularly between approximately 0.5 to 20 mg/kg.

The extracts and pharmaceutical compositions, in accordance with thepresent invention, may be advantageously administered in a single dailydose, or, alternatively, the total daily dose may be divided allowingadministration in two, three or more times per day.

In accordance with one embodiment of the present invention, a dose ofthe extract or pharmaceutical composition may be advantageouslyadministered for a period ranging from 1 day to several months.

For determined treatments, however, administration on alternate days isappropriate, either in a cyclical manner or not.

The dose regimen for the standardized extract or pharmaceuticalcomposition in accordance with the present invention naturally varies inaccordance with known factors, such as route of administration, species,age, sex, state of health, medical condition and weight of the patient,the nature and extent of the symptoms, the type of concurrent treatment,the intensity and degree of the illness, the frequency of the treatment,the patient's renal and hepatic functions, and the intended effect. Aphysician, dentist or veterinarian may determine and prescribe theefficient quantity of the medicine necessary to prevent, control ortreat a clinical condition of pain, inflammation or fever.

Uses

One of the aspects of the invention refers to the use of thestandardized extract for the preparation of a medicine for the treatmentof clinical conditions of pain, inflammation or fever.

The invention also refers to the use of the standardized extract for thepreparation of a medicine for the control of painful symptomologies ofnociceptive origin, in acute, sub-acute or chronic conditions.

The invention also refers to the use of a pharmaceutical composition forthe preparation of a medicine for the treatment of clinical conditionsof pain, inflammation or fever.

Lastly, the invention also refers to the use of a pharmaceuticalcomposition containing the standardized extract for the preparation of amedicine for the control of painful symptomologies of nociceptiveorigin, in acute, sub-acute or chronic conditions.

The following examples are more detailed descriptions of the preferredembodiments of the present invention. The procedures proposed and thedata presented constitute representative examples of applications of thepresent invention and should not be construed as indicating limits tothe uses of the product.

Example I Obtaining the Standardized Soft Extract from the Leaves ofAleurites moluccana

The leaves of A. moluccana are washed, dried, shredded and classified bypassing through a sieve, thus providing shredded leaves under 20 mm insize. The plant material is then submitted to extraction with ahydroalcoholic solution of ethanol/water in a proportion of 7:3. Thevolume used for extraction corresponded to 10 parts of extractor liquidto 1 of plant material. Extraction was performed at room temperature bythe maceration method, without agitation. After 5 days, the solution waspercolated and filtered through a paper filter. The filtrate wasconcentrated at a maximum temperature of 70° C., in a specificevaporator (Bernhauer), under vacuum (400 mmHg), until a non-alcoholicsoft extract was obtained with a total rate of solids between 20% and40%.

Obtaining the Dry Extract from the Leaves of Aleurites moluccana

The soft extract was obtained as described above. In sequence, avariable quantity ranging from 10 to 40% of drying adjuvant is added tothe extract in relation to the rate of solids of the final product. Themixture is dehydrated in a spray-drier using the centrifugal rotativedisc, at a pump input speed of 45 Hz, generating an output of 6.5 litresof concentrate/hour, at an input temperature of 160 to 180° C. andoutput temperature of 90 to 110° C.

Example II Qualitative Analysis of the Standardized Extracts ofAleurites moluccana

Qualitative Analysis of the Extracts by TLC

Thin layer chromatography (TLC) was used to analyse the constituents ofthe extracts of Aleurites moluccana, in accordance with the presentinvention, using alpha-amyrin and beta-amyrin, alpha-amirinone andbeta-amirinone, stigmasterol, beta-sitosterol, swertisin and2″-O-rhamnosylswertisin standards, isolated from the actual plant.

For the identification of the polar compounds, with emphasis onswertisin and 2″-O-rhamnosylswertisinisin, 1 g of the fraction of ethylacetate obtained from the extract EtOH:H₂O 9:1 of the leaves of A.moluccana, solubilised with methanol was weighed. The swertisin and2″-O-rhamnosylswertisin standards were then also weighed (1 mg each) andsolubilised with 0.1 ml of methanol. This was followed by theapplication of 10 μl, of each solution to a silica gel 60 plate (8 cmlong by 5.5 cm wide) and after drying, the plate was eluted withAcOEt:Acetone:H₂O (25:8:2). Elution was repeated three times. The platewas analysed in an UV chamber a subsequently revealed with a solution offerric chloride 3% in EtOH, calculating the retention factor (Rf) of thecompounds (Farmacopéia Brasileira, part II. 4. Ed. São Paulo: Atheneu,1996, 200p).

A comparative of the results revealed the presence of these compounds inboth the dry and soft extracts, as demonstrated by FIG. 1 (A).

For the identification of the non-polar compounds, with emphasis onalpha-, beta-amyrin, alpha-, beta-amirinone, stigmasterol,beta-sitosterol, 1 g of the fraction of dichloromethane obtained fromthe extract EtOH:H₂O 9:1 of the leaves of A. moluccana, solubilised withdichloromethane was weighed. The alpha-, beta-amyrin, alpha-,beta-amirinone, stigmasterol, beta-sitosterol, standards were then alsoweighed (1 mg each) and solubilised with 0.1 ml of dichloromethane. Thiswas followed by the application of 10 μL of each solution to a silicagel 60 plate and after drying, the plate was eluted with Hexane:AcOEt:(7:3). The plate was analysed in an UV chamber a subsequently revealedwith a solution of sulphuric anisaldehyde and heated to 60° C. on a hotplate, calculating the retention factor (Rf) of the compounds(Farmacopéia Brasileira, part II. 4. Ed. São Paulo: Atheneu, 1996,200p). A comparative of the results revealed the presence of thesecompounds in both extracts, as demonstrated by FIG. 1 (B).

Qualitative Analysis of the Extracts by Gas Chromatography (GC)

The analysis by gas chromatography was performed by a gas chromatographequipped with a flame ionisation detector (CG-FID), with a DB1 type(Agilent) column having an internal diameter of 30 m×0.25μ.

Test Solution: Exactly 10 mg of the fraction of dichloromethane obtainedfrom the extract EtOH:H₂O 9:1 solubilised with 0.3 ml of HPLC gradedichloromethane was first weighed. A micro-syringe was then used toinject 1 μL of the dichloromethane fraction.

Reference Solution: A micro-syringe was used to inject 1 μL of α andβ-amyrin (commercial reference standards—Sigma) at a concentration of 5mg/ml.

Chromatography Conditions: Column temperature at 200° C. for 2 min, thenrising at a rate of 10° C./min until attaining 290° C. and maintained atthis temperature for 40 minutes. Injector and detector temperatures of260° C. and 290° C., respectively. Split of 30 ml/min, with a septumpurge of 10 ml/min. Hydrogen used as the mobile gas at a flow of 1ml/min.

FIG. 23 shows the results obtained with the fraction of dichloromethanefrom Aleurites moluccana when assessed by Gas Chromatography (GC), usinga gas chromatograph equipped with a flame ionisation detector (CG-FID),with a DB1 type (Agilent) column having an internal diameter of 30m×0.25 mm. The test solution (fraction of dichloromethane from theextract EtOH:H₂O=9:1) was solubilised with 0.3 ml of HPLC gradedichloromethane.

A micro-syringe was used to inject 1 μL of the fraction ofdichloromethane. The reference solution (β-amyrin, Sigma standard) wasinjected in a concentration of 5 mg/ml, by means of a micro-syringe. Thechromatographic conditions were: Column temperature at 200° C. for 2min, then rising at a rate of 10° C./min until attaining 290° C. andmaintained at this temperature for 40 minutes. Injector and detectortemperatures of 260° C. and 290° C., respectively. Split of 30 ml/min,with a septum purge of 10 ml/min. Hydrogen used as the mobile gas at aflow of 1 ml/min. As shown by FIG. 23, the chromatogram of the dry andsoft extracts revealed the presence of the beta-amyrin standard (5.0mg/ml) at the retention time of 19.2±0.2 min and alpha-amyrin at theretention time of 20.23±0.2 min.

Example III Quantitative Analysis and Standardisation of the Extracts byHPLC

An analysis by High performance liquid chromatography (HPLC) wasperformed by dissolving the extracts of Aleurites moluccana initially inmethanol (20% of final volume), sonicating for 5 min, followed by theaddition of water acidified to pH 3.57 (20% of final volume). The volumewas then completed with a mixture of methanol/water acidified 50:50 at aconcentration of 1 to 2 mg/ml. After filtering the sample through a 0.22m membrane filter, 20 L of the solution was injected using a reversedphase analytical column, where the C18 groups are chemically linked tosilica, with a length of 10 to 25 cm and an internal diameter of 4 to 6mm. A photo diode array (PDA) type detector was used to monitor thechromatogrphic run at between 254 and 338 nm. Separation was performedusing a mobile phase linear gradient, initially composed ofmethanol:water acidified to pH 3.57: acetonitrile (20:15:65) at a flowof 0.5 to 1 ml/min, as described below:

Time MeOH ACN H₂O (pH 3.57) 0 20 15 65 10 30 15 55 20 10 15 75 25 20 1565

Pressure varied between 2.000 to 3.000 psi.

FIG. 5 (A) shows the HPLC chromatogram of the standard2″-O-rhamnosylswertisin purified by PTLC (AMSR170707) at 145 g/ml, in338 nm, which eluted in approximately 17 min, thus demonstrating successin purification of the marker, as indicated by the presence of a singlepeak on the chromatogram.

FIG. 5 (B) shows the absorption profile of the 2″-O-rhamnosylswertisinstandard, which is typical of flavonoids.

FIG. 2 (A) shows the chromatogram of the dry extract of Aleuritesmoluccana at 2 mg/ml, showing the peak resolution of the2″-O-rhamnosylswertisin marker (at approximately 17 min) compared to thefollowing peak (swertisin).

Apart from the coincidence in the retention time, the absorption profileat the UV of the peak that eluted in approximately 17 min on thechromatogram of the dry extract (FIG. 5 B) is identical to the2″-O-rhamnosylswertisin standard.

FIG. 2 (B) shows the chromatogram of the soft extract of Aleuritesmoluccana at 1.72 mg/ml and reveals a very similar profile to that seenfor the dry extract (FIG. 23 A), with the presence of a peak relating to2″-O-rhamnosylswertisin (at approximately 17 min) compared to thefollowing peak (swertisin).

It is therefore possible to confirm and quantify the presence of2″-O-rhamnosylswertisin in the extracts, observing a variation of 0.05to 15% of this marker in the extracts.

The method for quantifying 2″-O-rhamnosylswertisin in the extractys isvalid, in accordance with the official local regulation (Brazil.National Health Surveillance Agency, (Agência Nacional de VigilânciaSanitária). RE 899, 29^(th) May, 2003).

Example IV Process for the Isolation, Purification and Analysis of the2″-O-rhamnosylswertisin Marker from the Extract of Aleurites moluccanaUsing Preparative TLC

As the selected marker is not a commercial product, it was isolated andpurified from a fraction of ethyl acetate of the hydroalcoholic extractof A. moluccana.

Taking the soft extract, as described above, a liquid-liquid partitionwas performed, initially using dichloromethane and, later, ethylacetate. A fraction of acetyl previously concentrated until drying in arota-evaporator at 50° C. was submitted to open column chromatographyusing a silica gel 60 support, by taking 5 to 20 g of the fraction ofthe concentrated ethyl acetate prepared in pellet form through mixturewith silica gel 60. Chloroform was initially used as the mobile phasewith a gradual increase in polarity, using increasing proportions ofmethanol (from 9:1 to 5:5 of CHCl₃:methanol, ending with 100% methanol).The isolation of 2″-O-rhamnosylswertisin was monitored by TLC, asdescribed below (TLC profile of the extracts of Aleurites moluccana).

The final purification of the semi purified fractions obtained inaccordance with the above methodology was performed by means ofPreparative Thin Layer Chromatography (PTLC). The PTLC was performedusing a F254 silica gel 60 plate (1 mm thick, Merck), which received asolution of the semi purified fraction dissolved in methanol (10 mg in0.5 to 1.0 ml) and eluted with a mixture of chloroform:methanol 7:3. Theplate was then dried at room temperature before being analysed by UV andthe stain corresponding to 2″-O-rhamnosylswertisin (identified bypartial plate revelation using ferric chloride 3%) was removed andsolubilised with methanol, followed by filtration in paper to finallyobtain the marker. The filtrate containing the isolated marker was thendried at room temperature.

The referred standard obtained was then analysed by means of analyticTLC (in accordance with the methodology described in Example II above),HPLC (in accordance with the methodology described in Example IIIabove), infra-red spectrum with a KBr pellet, NMR H¹ and NMR C¹³, so asto ascertain purity and establish the standard batch number on aspecific form. The results of the analyses performed should be annexedto this form to render the process for obtaining the standard traceable.

The NMR C¹³, NMR H¹ and IR spectrums of the marker (batch AMSR170707),mentioned above, are to be found in the FIGS. 3 (A, B, C), 4 (A, B, C)and 6, respectively.

The elution profile by HPLC and the UV absorption profile (batchAMSR170707) are to be found in the FIGS. 5 A and B, respectively.

Example V Tests for Determining the Antinociceptive Activity of theExtract of A. moluccana in Acute Nociception Models

Assessment of the Antinociceptive Effect of the Extracts of Aleuritesmoluccana Using the Acetic Acid Induced Abdominal Contortion Model.

The experimental acetic acid induced abdominal contortion model allowsassessment of the antinociceptive activity of various substances thatact both at central and peripheral levels. Nociceptive response isinduces by the intraperitonal injection of acetic acid (0.6%) diluted insaline solution (0.9%). The abdominal contortions consist in thecontraction of the abdominal muscles combined with the extension of oneof the rear paws. The antinociception rate is assessed by the reductionin the number of abdominal contortions compared to the group of animalshaving received the vehicle (negative control) (Collier, H. D. J. etal., 1968, Br. J. Pharmacol., 32, 295-310).

Each analysis was performed using 8 to 10 male animals (Swiss Webstermice) weighing between 25 and 30 g. The dose was calculated individuallyfor each animal with the quantity in mg/kg (mg/1.000 g) of the substanceto be administrated being in accordance to its weight. The injectedvolume of the solutions followed the rule of 0.1 ml/10 g of the animal'sweight.

After treatment with the compounds and the injection of acetic acid, theanimals were placed under glass domes and observed individually, withthe number of abdominal contortions being cumulatively quantified duringa period of 20 minutes. The results were quantified according to thearithmetical average of the number of contortions observed in theanimals (all the animals of a specific treatment) followed by the ASEs(average standard errors).

Assessment of the Antinociceptive Effect of the Extracts of Aleuritesmoluccana Using the Formalin Induced Pain Model.

This model is more specific than the acetic acid induced abdominalcontortion model. It allows the assessment of two distinct types ofpain: the pain of neurogenic origin (direct stimulation of thenociceptive neurons) and of inflammatory origin, representing the tonicresponse to pain, accompanied by an inflammatory response related to therelease of chemical inflammation mediators. The nociceptive response wasinduced using 20 μL of formalin, that consists of a formaldehydesolution at 0.92% (2.5%, via intraplantar), injected in the dorsalregion of the animal's rear right paw with the vehicle being injected inthe dorsal region of the rear left paw. Immediately after the formalininjection, the animals were placed under a glass dome surrounded bymirrors in order to facilitate observation of the ensuing behaviour.Observation commenced immediately, initially timing the reaction to painduring the first 5 minutes after the formalin injection (correspondingto the period of neurogenic pain). Following an interval of 10 minutes,the licking times were recorded for a further 15 minutes (correspondingto the inflammatory pain). Total test time was of 30′.

At the end of this observation period, the animals were sacrificed bycervical displacement and the rear paws were removed at the tibiotarsalregion and weighed on an analytical balance to quantify the edemainduced by the formalin. The difference in weight (in mg) between theright paw (injected with formalin) and the left paw (injected withsaline) was considered the rate of edema. (Dubuisson, D. et al., 1977,Pain., 4, 161-174; Hunskaar, S. and Hole, K., 1987, Pain, 28, 343-355).Each analysis was performed using 8 to 10 male animals (Swiss Webstermice) weighing between 25 and 30 g.

The dose was calculated individually for each animal with the quantityin mg/kg (mg/1.000 g) of the substance to be administrated being inaccordance to its weight. The injected volume of the solutions followedthe rule of 0.1 ml/10 g of the animal's weight.

The animals were observed after the intraplantar injection of formalinfor a period of 30 minutes. The reaction time (time in which the animalremains licking the injected paw) is then quantified in each of thephases: phase I (neurogenic pain—5 min observation) and phase II(inflammatory pain—15 min observation).

Assessment of the Antinociceptive Effect of the Extracts of Aleuritesmoluccana Using the Hot Plate Model.

The hot plate model is normally used for verification of analgesicmedicines having an action mechanism that may involve the opioid route.The animals are placed on a hot plate (UGO BASILE, model L1912-06),programmed to a temperature of 56° C. (±1) and the time (in seconds)that each animal takes to lick, bite or lift its paws is consideredindicative of pain. The maximum permissible time for the animals toremain on the plate is 30 seconds in order to prevent tissue damagecaused by the heat. In this model, the animals are pre-selected 24 hoursbefore the test so as to ascertain their pain threshold without beingsubmitted to any treatment. The animals with a pain threshold under 10 s(observed in the pre-test) were submitted to the test. A positivecontrol for the test was also performed, with the animals beingsubmitted to treatment with morphine (5 mg/kg) administeredsubcutaneously (Souza, M. M. et. al. Métodos de avaliação de atividadebiológica de produtos naturais e sintéticos. In Cechinel Filho, V.,Bresolin, T. M. B. Ciências Farmacêuticas: Contribuição desenvolvimentode novos fármacos e medicamentos, 2003, 108-166).

Each analysis was performed using 8 to 10 male animals (Swiss Webstermice) weighing between 25 and 30 g. The results were quantifiedaccording to the arithmetical average of the reaction time in secondsafter being placed on the hot plate (the time span before the animalspresented thermal sensitivity which was expressed by licking the rearpaws and an attempt to escape the pain by rearing), followed by the ASEs(average standard errors).

Statistical Analysis:

Parametric statistical tests were performed on the models describedabove. The results were submitted to ANOVA variance analysis followed byappropriate post hoc tests. In the case of the pain and inflammationmodels, the results were presented as average±average standard error(ASEs), except for DI50 (the dose that reduces response by 50% inrelation to the control group), which was presented as a geometricaverage accompanied by its respective reliability limit at 95% level.The statistical analyses of the results were performed by means ofvariance analysis followed by the multiple comparison test using theDunnett method, when appropriate. Values of p<0.05 were considered toindicate significance. The DI50 was estimated by means of individualexperiments using GraphPad Instat statistical software.

Example VI Tests for Determining the Anti-Inflammatory Activity of theExtract of A. moluccana.

Assessment of the Anti-Inflammatory Effect of the Dry Extract ofAleurites moluccana Using the Phlogistic Agent Induced Edema of the PawModel.

The edema of the paw pharmacological model is not considered as aninflammation model per se, but rather as a model of antiedematogenicactivity since it merely assesses one of the parameters of theinflammatory process, namely, the formation of the edema. The phlogisticagent used in the experiment is generally carragenine. Several stages ofthe process may be assessed during the “inflammation” induced by thisagent. The histamine, serotonin and substance P mediators are releasedbetween 0 and 2 hours after administration of the carragenine,prostaglandins, nitric oxide and several tachykinins are released at thepeak of the maximum response to the inflammation (following the fourthhour of the experiment).

In this manner, it is possible to ascertain in which phase of theinflammatory process a compound acts, and, later, induce the edema withthat specific mediator. Therefore, it is possible to characterise boththe antiedematogenic effect and also the mechanism of action of thecompound undergoing study, or, in other words, which mediators of theinflammatory process are supposedly inhibited by the compound.

Edema of the Paw Induced by Carragenine

During the trials, the animals received intraplantar injections of thephlogistic agent, carragenine, at a concentration of 300 ug/paw dilutedin saline solution in the right paw (0.025 ml/paw), and physiologicalsolution in the left paw (0.025 ml/paw). The several concentrations ofthe dry extract and the negative control (physiological solution) wereadministered orally (0.3 ml/100 g of the animal's weight) 1 hour beforethe injection with carragenine. The positive control (dexamethazone 0.5mg/kg) was administered subcutaneously 4 hours before. The edema of thepaw was measured plethysmometrically (plethysmometer Ugobasile, Italy)at time intervals of 0.5, 1, 2, 3 and 4 hours after injection of thephlogistic agent. The variance in paw volume was expressed in ml and thedifference between the volume of the right and left paws is consideredas being the edema rate (Souza, M. M. et al. (2003)).

Edema of the Paw Induced by Bradykinin

During the trials, the animals received intraplantar injections of thephlogistic agent, bradykinin, at a concentration of 300 ug/paw dilutedin saline solution in the right paw (0.025 ml/paw), and physiologicalsolution in the left paw (0.025 ml/paw). The several concentrations ofthe dry extract and the negative control (physiological solution) wereadministered orally (0.3 ml/100 g of the animal's weight) 1 hour beforethe injection with bradykinin. The positive control (dexamethazone 0.5mg/kg) was administered subcutaneously 4 hours before. The edema of thepaw was measured plethysmometrically (plethysmometer Ugobasile, Italy)at time intervals of 0.5, 1 and 2 hours after injection of thephlogistic agent. The variance in paw volume was expressed in ml and thedifference between the volume of the right and left paws is consideredas being the edema rate (Souza, M. M. et al. (2003)).

Edema of the Paw Induced by Histamine

During the trials, the animals received intraplantar injections of thephlogistic agent, histamine, at a concentration of 0.19/paw diluted insaline solution in the right paw (0.025 ml/paw), and physiologicalsolution in the left paw (0.025 ml/paw). The several concentrations ofthe dry extract and the negative control (physiological solution) wereadministered orally (0.3 ml/100 g of the animal's weight) 1 hour beforethe injection with histamine. The positive control (dexamethazone 0.5mg/kg) was administered subcutaneously 4 hours earlier. The edema of thepaw was measured plethysmometrically (plethysmometer Ugobasile, Italy)at time intervals of 0.5, 1 and 2 hours after injection of thephlogistic agent. The variance in paw volume was expressed in ml and thedifference between the volume of the right and left paws is consideredas being the edema rate (Souza, M. M. et al. (2003)).

Each of the analyses described above required 8 to 10 male animals(Wistar Rats) weighing between 200 and 300 g.

The results were quantified through the arithmetical average obtainedfor the edemas of the paws from each treatment group, followed by theSEAs (standard errors of average), across the measurement times duringeach experiment: carragenine (6 measurements in 4 hours of experiment),bradykinin and histamine (3 measurements in 2 hours of experiments).

Assessment of the Anti-Inflammatory Effect of the Dry Extract ofAleurites moluccana Through the Pleurisy Induced by the IntrapleuralInjection of Phlogistic Agents Model

Among the animal models available for assessing the anti-inflammatoryactivity of substances, the pleurisy model is considered to be one ofthe more complete because the drained fluids from the pleural cavityallow the analysis and quantification of the cellular and humoralcomponents of the inflammation without resorting to meticulousextraction and quantification procedures. (Saleh T. S. F., Calixto, J.B. Medeiros, Y. S., 1997, Eur J. Pharmacol., 331, 43-52). Pleurisy maybe induced in animals by phlogistic agents such as carragenine,bradykinin, Substance P, prostaglandin E₂ etc. and, as before in theedema of the paw model, the process is first induced using carragenin.The animals were treated with different concentrations (125, 250, 500mg/kg) of the dry extract of A. moluccana, and the vehicle, (salinesolution) administered orally. They then received an endovenousinjection (0.2 ml) of Evans blue solution (25 mg/Kg). The phlogisticagent (carragenine 1.0%, Substance P (20 nmol), and bradykinin (10nmol), histamine (100 μg/cavity) was administered intrapleurally onehour after injection of the above treatments. This provides a grouptreated with the extract and with the phlogistic agent, a negativecontrol group (pre-treated with saline solution v.o. and receivingsaline solution i.pL.) and another control group (pre-treated withsaline solution v.o. and receiving the phlogistic agent i.pL.) Fourhours after treatment with the phlogistic agent, the animals weresacrificed and their abdominal region was opened rupturing thediaphragm. The pleural cavity was rinsed with a heparinised (20 UI/ml)saline solution (1.0 ml). The drained pleural fluid was then collectedwith an aliquot (10 μL) of the latter being added to a Turk solution (2%acetic acid) at a proportion of 1:20. Part of this material (10 μL) wastransferred to a Neubauer chamber for a total cell count. The remainingpleural wash was centrifuged (10.000 rpm×10 min). The supernatant wasremoved and used for an ELISA reading (620 nm) with the purpose ofmeasuring the plasma outflow (edema) after the inflammatory stimulus.The pellet was resuspended with 100 μL egg albumin (3%) and 10 μL wasadded to a slide covered in perforated filter paper which was then leftto dry before staining using the May-Grunwald-Giemsa method fordifferential cell count by microscopy (enlarged 100 times).

Assessment of the Anti-Inflammatory Effect of the Extracts of Aleuritesmoluccana Using the Croton Oil Induced Ear Edema Model

The ear edema model is specific and broadly used to research theactivity of anti-inflammatory agents of topical use. The irritant agentused is croton oil and the extent of the edema is measured by thedifference in the thickness of the ear before and after treatment(Montello, M. S. A. G., Efeito da terapia com laser de baixa potência(HeNe e AsGa) na dermatite induzida por óleo de cróton na orelha decamundongo. São José dos Campos, 2002. 82p. Dissertação Mestrado emEngenharia Biomédica—Instituto de pesquisa edesenvolvimento—Universidade do Vale do Paraíba).

A “basal” measurement of the ear thickness of the animal was performed,immediately followed by application of the control substance or theextract to the right inner ear. After 30 minutes, croton oil was appliedto the outer part of the same ear. The ear was then measured again 4hours after treatment with croton oil.

Each analysis was performed using 8 to 10 male animals (Swiss Webstermice) weighing between 25 and 30 g. The results were quantifiedaccording to the arithmetical average obtained for the ear edemas foreach treatment group over the measurement times during the experiment (2measurements in 6 hours of experiment run time) followed by the ASEs(average standard errors).

Assessment of the Antipyretic Effect of the Extracts of Aleuritesmoluccana Using the LPS Induced Hyperthermia Model in Rats.

Antipyretic properties are also observed in NSAIDs and, the assessmentof this pharmacological property is also important when assessinganalgesic and anti-inflammatory substances. The onset of the febrileprocess involves mediators such as prostaglandins and interleukins,amongst others, whose activities are associated to the processes ofanalgesia and inflammation. Generally, the pharmaceuticals thatinterfere with the activities of these mediators exhibit all threepharmacologic properties. The LPS induced hyperthermia model was used toverify the antipyretic effect of the soft extract of A. moluccana.Groups of animals were treated the soft extract of the plant in twodoses (5.0 and 10 mg/kg), with a positive control (sodium dipyrone,mg/kg) and/or negative (vehicle) by intraperitoneal route. The basaltemperatures of all groups were recorded after 60 minutes, followingwhich the groups (treated and control) received an intraperitonealinjection of LPS (10 μg/Kg, diluted in physiological solution). Thetemperatures were verified by carefully inserting veterinarythermometers in the rectal canal of each animal and maintaining it therefor approximately 1 minute. The temperatures were recorded 40 minutesafter injecting the LPS and at intervals of 40 minutes thereon for aperiod of 7 hours and 20 consecutive minutes, or, in other words, at 40,80, 120, 160, 200, 240, 280, 320, 400 and 1.440 minutes. Results wereexpressed as the difference between the final temperatures and the basaltemperature as described by Roth and De Souza (Roth, J. De-Souza,G.E.P., 2001, Brazilian Journal of Medicinal and Biological Research 34,301-314).

Male Wistar rats (250 to 300 g) were pre-treated with A. moluccana (5 to10 mg/Kg), and/or vehicle and dipyrone (10 mg/Kg) and, after 1 hour,submitted to the LPS induced fever model (10 μg/Kg). The temperaturevariations were verified at intervals of 40 minutes, over a period of 8hours. Possible alterations to the gastric mucosa of the animals causedby the plant were also assessed by means of the above treatment but withthe additional of indomethacin (30 mg/Kg).

Statistical Analysis:

Statistical analysis of the data was performed by means of one-way ANOVAvariance analysis followed by the Dunnett test. Values of p<0.05 wereconsidered to indicate significance. The DI50 (the dose that reducesresponse by 50% in relation to the control group), which was presentedas a geometric average accompanied by its respective reliability limitat 95% level and was estimated by means of individual experiments by thelinear regression method using GraphPad® software. The DI50 calculationis used for potency comparison between the test-substance andtraditional pharmaceuticals. The inhibition percentages were stated asthe average±the average standard error of the difference (in percentage)for each individual experiment in relation to the corresponding controlgroup. EXAMPLE VII

Tests for Determining the Anti-Hypernociceptive Activity of the Extractof A. moluccana in Persistent Pain models.

Assessment of the Effect of the Dry Extract of Aleurites moluccana onMechanical Hypernociception Induced by the Intraplantar Injection ofCarragenine.

The mice had inflammatory hyper-nociception induced by an intraplantarinjection of 50 μl of carragenine (300 ug/site) on the plantar surfaceof the right rear foot. This dose is capable of producing an edema,hyper-nociception and significant swelling of the injected paw but,however, the animals continue to present normal behaviour. (De Campos etal., 1996, Eur J. Pharmacol., 316, 2-3, 277-86; Bortolanza et al., 2002,Eur. J. Pharmacol., 453, 2-3, 203-8; Quintão et al., 2005, AnesthAnalg., 101, 6, 1763-9; Quintão et al., 2006, Neuropharmacol., 50, 5,614-620). The animals were initially pre-treated with the dry extract ofA. moluccana, administered orally, (125-500 mg/kg; v.o.), 1 h prior toinducing hypernociception. The animals then received an intraplantarinjection of carragenine (300 μg/paw) and were assessed for mechanicalhypernociception using the von Frey filament, as described below in item(e), at different times (1, 3, 4, 6, 24 and 48 h). The control group wastreated with the vehicle (10 ml/kg) used for dilution of the extract(saline 0.9%).

Assessment of the Effect of the Dry Extract of Aleurites moluccana onMechanical Hypernociception Induced by the Intraplantar Injection ofProstaglandin E₂ (PGE₂).

The animals were initially pre-treated with the dry extract of A.moluccana, administered orally, (125-500 mg/kg; v.o.), 1 h prior toinducing hypernociception. At the end of the treatment time, the animalsreceived injections i.pl. of PGE₂ (0.1 nmol/paw) (Kassuya et al., 2007,Br J. Pharmacol., 150(6), 727-737) after which they were assessed formechanical hypernociception using the von Frey filament 0.6 g, asdescribed below in item (e), at different times (0.5, 1, 2, 4, 6 and 24h). The control group was treated with the vehicle (10 ml/kg) used fordilution of the extract (saline 0.9%).

Assessment of the Preventive and Curative Effect of the Dry Extract ofAleurites moluccana on Mechanical Hypernociception Induced by theIntraplantar Injection of the Complete Freund Adjuvant (CFA).

In order to induce a persistent inflammatory response, the animalsreceived an injection i.pl of 20 μl of complete Freund adjuvant (CFA; 1mg/ml of mycobacterium tuberculosis bacillum inactivated by heat; witheach mililitre (ml) of the vehicle containing 0.85 ml of paraffinoil+0.15 ml of manide monooleate) on the plantar surface of the rearright paw (Cao et al., 1998, Nature, 392, 390-394; Ferreira et al.,2001, Neuropharmacol., 41, 8, 1006-1012). Mechanical and thermalhypernociception were assessed using the von Frey filament 0.6 g, asdescribed in below in item (e). In order to assess the preventive effecton mechanical hypernociception, the animals were initially pre-treatedwith the dry extract of A. moluccana, administered orally, (125-500mg/kg) or the vehicle (10 ml/kg). After lh of treatment, the animalsreceived an injection i.pl CFA, and mechanical hypernociception wasassessed at different time intervals (1, 2, 4, 6, 8, 12, 24 and 48 h).

With the intent of verifying the curative treatment, the animalsreceived an intraplantar injection of CFA and after 24 h were thentreated orally with the dry extract of A. moluccana (125-500 mg/kg) orvehicle (10 ml/kg), twice a day (12 in 12 h) for 5 consecutive days withthe mechanical hypernociception of the ipsilateral and contralateralpaws to the injection of CFA being assessed 6 h after administration ofthe first daily dose, as described above. In order to investigate theextent of the antinociceptive effect of A. moluccana in the experimentsusing CFA, the treatment was interrupted 5 days after administration ofthe first dose and then renewed after 2 days to investigate any possibletolerance development.

Assessment of the Preventive and Curative Effect of the Dry Extract ofAleurites moluccana on Mechanical Hypernociception Induced by thePartial Constriction of the Sciatic Nerve (PCSN).

The procedure used was similar to the one described for rats by Seltzeret al. (1990) and modified for use with mice by Mamberg and Basbaum(1998). The mice were anaesthetised with chloral hydrate 7% (8 ml/kg;i.p.).

Constriction of the sciatic nerve was acheived tying ⅓ to ½ of thedorsal part of the sciatic nerve with 8-0 stitching thread (Ethicon®). Agroup of animals had the sciatic nerve exposed without, however, anytying process (false operated group). On the fourth post-operatory day,the animals were assessed for mechanical hypernociception using the vonFrey monofilament, as described below in item (e) and were then treatedorally with the dry extract of A. moluccana (125-500 mg/kg) or vehicle(10 ml/kg), twice a day (12 in 12 h) for 5 consecutive days with themechanical hypernociception being assessed 6 h after administration ofthe first daily dose.

Analysis of the Mechanical Threshold Using the Von Frey FilamentTechnique.

To assess the mechanical hypernociception, the animals subjected to thecarragenin, CFA or PCSN induced hypernociception models were placedsingly in transparent acrylic individual compartments (9×7×11 cm)located on a raised wireframe platform to allow access to the plantarsurface of the rear paws. The animals were acclimatised for at least 30minutes before initiating the behavioural tests. The frequency of thewithdrawal response was obtained using 10 applications (each lasting 1s) of the von Frey filament 0.6 g (VFH, Stoelting, Chicago, USA). Thestimuli were applied to the plantar surface of the animal's rear rightpaw (Quintão et al., 2005, Anesth Analg., 101, 6, 1763-9; Quintão etal., 2006, Neuropharmacol., 50, 5, 614-620). In order to determine thebasal mechanical threshold (B), all the groups of animals were submittedto prior assessment a then newly assessed at different times after thecarragenine or CFA injections, or after the PCSN.

Statistical Analysis:

The statistical analysis of the data was performed by means of thevariance analysis (two-way ANOVA) followed by the Bonferroni test.Values of p<0.05 were considered to indicate significance. The DI50s(the dose that reduces response by 50% in relation to the control group)were presented with geometric averages accompanied by their respectivereliability limit at a 95% level and were estimated by means ofindividual experiments using the linear regression method of theGraphpad® software. The DI50 calculation is used for potency comparisonbetween the test-substance and traditional pharmaceuticals. Theinhibition percentages were stated as the average±the average standarderror of the difference (in percentage) between the areas under thecurves plotted for each individual experiment in relation to thecorresponding control group.

Example VIII Assessment of Pharmacological Effect of the Dry Extract ofA. Moluccana Neuropathic Pain Test in Mice by

The pharmacological assayes were performed with Swiss mice (n=8 to 10animals), female, weighing between 25 to g, maintained under controlledtemperature and lighting, with feed and water “ad libitum”, exceptduring the experiment period. Each experiment used from 8 to 10 animalsin each treatment group and after weighing, these were identified withsequential numbers using a marker-pen.

Partial Sciatic Nerve Constriction (PSNC) Inducement

The procedure used was similar to the one described for rats by Seltzeret al. (1990) and modified for use with mice by Mamberg and Basbaum(1998). The mice were anaesthetised with 7% chloral hydrate (8 ml/kg;i.p.). Constriction of the sciatic nerve was acheived tying ⅓ to ½ ofthe dorsal part of the sciatic nerve with 8-0 stitching thread(Ethicon®). A group of animals had the sciatic nerve exposed without,however, any tying process (false operated group). On the fourthpost-operatory day, the animals were assessed for mechanicalhypernociception using the von Frey monofilament, as described below initem (e) and were then treated orally with the dry extract of A.moluccana (125-500 mg/kg) or vehicle (10 ml/kg), twice a day (12 in 12h) for 5 consecutive days with the mechanical hypernociception beingassessed 6 h after administration of the first daily dose.

Analysis of the Mechanical Threshold Using the Von Frey FilamentTechnique.

To assess the mechanical hypernociception, the animals subjected to PCSNinduced hypernociception models were placed singly in transparentacrylic individual compartments (9×7×11 cm) located on a raisedwireframe platform to allow access to the plantar surface of the rearpaws. The animals were acclimatised for at least 30 minutes beforeinitiating the behavioural tests. The frequency of the retrievalresponse was obtained using 10 applications (each lasting 1 s) of thevon Frey filament 0.6 g (VFH, Stoelting, Chicago, USA). The stimuli wereapplied to the plantar surface of the animal's rear right paw (Quintãoet al., 2005, Anesth Analg., 101, 6, 1763-9; Quintão et al., 2006,Neuropharmacol., 50, 5, 614-620). In order to determine the basalmechanical threshold (B), all the groups of animals were submitted toprior assessment a then newly assessed at different times after thePCSN.

The statistical analysis of the data was performed by means of thevariance analysis (two-way ANOVA) followed by the Bonferroni test.Values of p<0.05 were considered to indicate significance.

Effect of the Dry Extract of Aleurites moluccana on MechanicalHypernociception Induced by the Partial Constriction of the SciaticNerve

The oral treatment of the dry extract of Aleurites moluccana (industrialbatch) when administered in doses of 125 to 500 mg/kg, was capable ofsignificantly reducing the mechanical hypernociception induced by PSNC.This inhibition was significant for up to 8 days, when the treatment wasinterrupted, as seen in FIG. 24.

FIG. 24 shows the effect of the treatment using the dry extract ofAleurites moluccana (125 to 500 mg/kg, administered orally) formechanical hypernociception induced by PSNC. Each point represents theaverage of 6 to 8 animals and the vertical bars indicate the ASEs. Theresults differ significantly in relation to the control group (C),*P<0.05, **P<0.01 e***<0.001.

Example IX Acute Toxicity Trial of the Extract of Aleurites moluccana

This study refers to an estimate and preliminary assessment of the toxicproperties of a test-substance, providing information relating to therisks to health resulting from an exposure of short duration by thechosen route of administration. The acute toxicity further serves as abasis for determining a dose regimen for the research relating tosubchronic, chronic and repeated dose toxicity, as well as providinginitial information concerning the toxic action mode of thetest-substance.

The experiment used 10 male mice and 10 female mice, divided in 4groups: a) male group treated with the vehicle (saline solution), b)male group treated with the soft extract of A. moluccana (5000 mg/Kg);c) female group treated with the vehicle, and d) female group treatedwith the extract (5000 mg/Kg). Food was withheld for 6 hours, followingwhich the animals received a force-feeding treatment and wereimmediately transferred to individual boxes for observation using thehippocratic test as described by Brito (1994) (Brito, A. S. Manual deEnsaios toxicológicos in vivo. Campinas: UNICAMP, 199, 41-121). Theanimals were observed individually and systematically for 24 hours aftertreatment for intervals of (0 to 10 min, 30 min, 60 min, 180 min and1.440 min). The supply of water and feed was restricted during the first4 hours of observation. The behaviours and/or manifestations to be notedwere: piloerection, salivation, cage corner movement, tremors,tachycardia, irritability, miction, defecation, ptosis, convulsions andthe number of deaths. Special attention was given to symptoms such as:tremors, convulsions, salivation, diarrhoea, irritability, lethargy andcoma. In the course of the experiment, any deaths and their times ofoccurrence are registered and, whenever possible, the causes areinvestigated. At the end of the experiment the animals were weighed andlater sacrificed. The results were quantified on average in terms of theappearance of the behavioural response being observed at the specifictime of observation. For example, if only 3 out of the 5 animals of theexperiment were observed to defecate, the table will record ⅗. Thelargest dose used was of 5000 mg and as no deaths were verified, a DL50(consisting of a value statistically derived from the administration ofa single dose capable of causing the death of 50% of the animals of anexperiment) couldn't be calculated. The test-substance with a DL50 over5 g has a great possibility of not exhibiting toxic effects.

The statistical analysis of the data was performed by means of thevariance analysis followed by the T test. Values of p<0.05 wereconsidered to indicate significance.

Example X

An example of a pharmaceutical composition in the form of a coated pillmay be obtained by the following formulation 1:

Ingredients Contribution % Code Nucleus: Dry extract of Aleurites 40%  ASpray-dried lactose 28%  B Crospolyvinylpirrolidone 10%  CMicrocrystalline cellulose 8 D Ascorbic acid 1% E Sodic croscarmelose 2%F Magnesium stearate 1.5%   G Coating: Eudragit ® 7% H Propylenoglycol1% I Titanium Dioxide 0.07%   J Yellow iron oxide 0.01%   KTriethylcitrate 0.20%   L TOTAL 100% 

Preparation Method

The nucleus is prepared by first individually weighing and sieving thecomponents. Following which, the components A, B, C and D are mixed in a“v” type mixer for 20 minutes. Components F and G are then added andmixed for a further 5 minutes. In sequence, the mixture is submitted todirect compression to form the compressed nuclei. The latter are thencoated with a solution containing components H, I, J, K and L byatomisation (aspersion) to form a coating film.

Example XI

An example of a pharmaceutical composition in the form of a capsule maybe obtained by the following formulation 2:

Ingredients Contribution % Code Dried extract of Aleurites 30% AMannitol 50% B Microcrystalline cellulose 10% C Ascorbic acid  1% DPregelatinised starch  8% E Talcum  1% F TOTAL 100% 

Preparation Method

The capsules are prepared by first weighing, mixing and sieving thecomponents A, B, C and D and in sequence, the mixture is submitted to awet granulation process in adequate equipment by gradually adding asolution containing the agglutinat E. Following which, the granules areimmediately submitted to drying in an oven at 30 a 40° C., untilattaining a humidity rate of approximately 2%. In sequence, the drygranules are submitted to classification and mixed with lubricant F forminutes. The classified dry granules are then encapsulated.

Example XII

An example of a pharmaceutical composition in the form of a suspensionmay be obtained by the following formulation 3:

Components Contribution % Code Dry extract of Aleurites   30% A Citricacid 1.00% B Sodium citrate 1.00% C Carboxymethylcellulose 5.00% DGlycerine 3.00% E Propylenoglycol 5.00% F Sorbitol 40.00%  G Essence0.01% H Methylparaben 0.10% I Propylparaben 0.01% J Deionised water QSP100.0% K TOTAL 100%

Preparation Method

The suspension is prepared by first weighing and mixing components A andF in a homogenising tank. In sequence, part of component K and theremaining components are gradually added to the mixture under constantagitation. The remainder of component K is then added to make up theintended volume. Finally, the mixture is submitted to mechanicalagitation until complete homogenisation of the solution is attained.

Example XIII

An example of a pharmaceutical composition in the form of a syrup may beobtained by the following formulation 4:

Components Contribution % Code Soft extract of Aleurites   30% A Citricacid 1.00% B Sodium citrate 1.00% C Essence 0.10% D Methylparaben 0.10%E Propylparaben 0.01% F Glycerine 5.00% G Colorant 0.01% H Sucrose50.00%  I Deionised water QSP 100.0 J TOTAL 100%

Preparation Method

The suspension is prepared by first weighing and mixing components A andF in a homogenising tank. In sequence, part of component K and theremaining components are gradually added to the mixture under constantagitation. The remainder of component K is then added to make up theintended volume. Finally, the mixture is submitted to mechanicalagitation until complete homogenisation of the solution is attained.

All publications mentioned in the above specification, and citedreferences therein are incorporated by reference. Several modificationsand changes of process, extracts, compositions, methods and usesdescribed in the present invention will be obvious to be practicedwithin the scope of this invention by those skilled in the art.

1-41. (canceled)
 42. A standardized extract of at least one part of aplant of the genus Aleurites, wherein said extract is distinguished byat least one of the aspects selected from the group consisting of: (i)chromatograms by high performance liquid chromatography (HPLC) specificto the soft extract and dry extract of the said plant of genusAleurites, (ii) NMR 0¹³ spectrums of the 2″-O-rhamnosylswertisin markermeasured at 300 MHz in deuterated methanol, (iii) NMR H¹ spectrums ofthe 2″-O-rhamnosylswertisin marker measured at 300 MHz in deuteratedmethanol, (iv) chromatograms by high efficiency liquid chromatography ofthe 2″-O-rhamnosylswertisin standardized purified by preparativethin-layer chromatography and (v) infra-red spectrum of the2″-O-rhamnosylswertisin marker.
 43. The extract according to claim 42,wherein said HPLC chromatograms specific for soft extract and dryextract are represented in, but not limited to, FIG.
 2. 44. The extractaccording to claim 42, wherein said NMR C¹³ spectrums are representedin, but not limited to, FIG.
 3. 45. The extract according to claim 42,wherein said NMR H¹ spectrums are represented in, but not limited to,FIG.
 4. 46. The extract according to claim 42, wherein said HPLCchromatograms of the 2″-O-rhamnosylswertisin standard are representedin, but not limited to, FIG.
 5. 47. The extract according to claim 42,wherein said infra-red spectrum of the 2″-O-rhamnosylswertisin markerare represented in, but not limited to, FIG.
 6. 48. The extractaccording to claim 42, comprising at least one active ingredientselected from the group consisting of alpha-amyrin, beta-amyrin,alpha-amirinone, beta-amirinone, swertisin, with the said extract beingstandardized in relation to its marker 2″-O-rhamnosylswertisin.
 49. Theextract according to claim 42, wherein said standardized extract ofAleurites moluccana contains a ratio of 2″-O-rhamnosylswertisin in therange of 0.05 to 15%.
 50. The extract according to claim 42, comprisinganalgesic, anti-inflammatory and antipyretic properties, in mammals. 51.A pharmaceutical composition comprising: (i) a pharmaceuticallyeffective quantity of the standardized extract in accordance with claim42 and (ii) at least one pharmaceutically acceptable carrier.
 52. Thepharmaceutical composition according to claim 51, wherein saidstandardized extract is present in a quantity that represents between 5and 90% of the composition's total weight.
 53. The pharmaceuticalcomposition according to claim 52, wherein said standardized extract ispresent in a quantity that represents between 20 and 80% of thecomposition's total weight.
 54. The pharmaceutical composition accordingto claim 51, comprising, additionally, at least one pharmaceuticallyactive substance selected from the group consisting of synthetic andsemi-synthetic substances, biological molecules, vitamins and otherderivative substances of plant origin.
 55. The pharmaceuticalcomposition according to claim 51, comprising pharmaceuticallyappropriate forms for oral, topical, intravenous, subcutaneous,intramuscular, intravaginal and/or rectal administration.
 56. Thepharmaceutical composition according to claim 55, wherein saidpharmaceutical composition is selected from among tablets, capsules(either soft or hard), pills, powders, granules, simple pills, coatedpills, chewable tablets, effervescent tablets, sublingual pills,controlled release pills, dragees, globules, elixirs, suspensions,syrups and emulsions, each of which may contain immediate, controlled,prolonged or retarded release formulations, ointments, unguents, creams,emulsions, gels, solutions, pastes, aerosols, transdermic, bolus orinfusion, subcutaneous or intramuscular systems, suppositories, ovules,ointments, creams and the similar.
 57. A process for preparing thestandardized extract according to claim 42, comprising the use of atleast one part of a plant of the genus Aleurites, and comprising thestages of: (i) collecting, drying and subdividing the plant material;(ii) pre-extracting; (iii) extracting with extractor solvent; (iv)filtrating the extract; (v) concentrating the extract; (vi)pasteurising; (vii) drying; and (viii) proceeding qualitative andquantitative analysis (standardisation) of the extract, with saidstandardized extract being distinguished by the 2″-O-rhamnosylswertisinmarker.
 58. The process according to claim 57, wherein said plant isselected from the group consisting of A. trisperma, A. cordata, A.montana, A. fordii, A. montance, A. rockinghamensis and A. moluccana.59. The process according to claim 57, wherein said plant of the genusAleurites is Aleurites moluccana L. Wild.
 60. The process according toclaim 59, wherein said plant material comprises at least the leaves ofAleurites moluccana L. Wild.
 61. The process according to claim 57,wherein the collecting stage of the plant material occurs in dryweather.
 62. The process according to claim 57, wherein thepre-extraction stage is performed by agitation in an alcoholic medium atroom temperature.
 63. The process according to claim 57, wherein theextractor solvent used for the extraction stage is selected from a groupconsisting of, but not limited to, water, methanol, ethanol, propanol,isopropanol, propyleneglycol, acid solution, ethyl acetate,dichloromethane, chloroform, hexane, glycerine, acetone, petroleumether, supercritical fluid, and similar or a combination thereof. 64.The process according to claim 63, wherein said extractor solvent is ahydro-alcoholic solution of water:ethanol in which the ratio of waterdoes not exceed the ratio of alcohol.
 65. The process according to claim63, wherein said extractor solvent is a hydro-alcoholic solution ofwater:ethanol at a ratio selected between 2:8, 3:7, 4:6 and 1:1.
 66. Theprocess according to claim 63, wherein said extractor solvent is ahydro-alcoholic solution of water:ethanol at a ratio of 3:7.
 67. Theprocess according to claim 57, wherein the ratio of extractorsolvent:plant material used in the extraction stage varies betweenapproximately 1:1 and 20:1.
 68. The process according to claim 57,wherein said ratio of extractor solvent:plant material is of 10:1. 69.The process according to claim 57, wherein the pasteurisation stage isperformed at a temperature of approximately 95° C., for approximately 3minutes.
 70. The process according to claim 57, wherein the stage forconcentrating the extract provides a concentration of approximately 20%to 50% of solids in the extract and is capable of completely eliminatingthe alcohol.
 71. The process according to claim 57, wherein the dryingstage is performed in conditions appropriate to maintain thequantitative and qualitative characteristics of the extract.
 72. Theprocess according to claim 71, wherein the drying stage is performed bythe spray-drying technique, in the presence of a drying adjuvant. 73.The process according to claim 72, wherein the drying adjuvant isselected from a group consisting colloidal silicon dioxide, modifiedcassaya starch, tricalcium phosphate, maltodextrin, cyclodextrins,microcrystalline cellulose, lactose or a combination thereof, added in aproportion of approximately 10 to 40% in relation to the ratio of totalsolids in the extract.
 74. The process according to claim 57, optionallycomprising a depigmentation stage of the extract, using a procedureselected from the group consisting of active charcoal treatment,adsorption resin treatment or membrane ultrafiltration.
 75. The processaccording to claim 57, optionally comprising a stabilisation stage ofthe extract, in the presence of a stabilising agent, with the saidstabilising agent being selected from the group consisting of derivatesof thyol, ascorbic acid and its derivatives, cysteine, glutathione, or acombination of the above and the similar.
 76. A method of treating pain,inflammation and/or fever in a mammal comprising administering to themammal an effective quantity of the standardized extract according toclaim
 42. 77. A method of treating pain, inflammation and/or fever in amammal comprising administering to the mammal at least one effectivequantity of the pharmaceutical composition according to claim
 51. 78.The method according to claim 74, wherein the dose to be administeredprovides 0.1 to 50 mg of the standardized extract per kilogram of thepatient's body weight.
 79. A method of treating pain, inflammationand/or fever in a mammal comprising administering to the mammal aneffective quantity of 2″-O-rhamnosylswertisin, isolated and purified,from the extract according to claim 42.